Compositions and methods for modifying blood cell carbohydrates

ABSTRACT

This invention relates to enzymatic removal of type A and B antigens from blood group A, B, and AB reactive cells in blood products, and thereby converting these to non-A and non-B reactive cells. The invention further relates to using unique αN-acetylgalactosaminidases and α-galactosidases with superior kinetic properties for removing the immunodominant monosaccharides of the blood group A and B antigens and improved performance in enzymatic conversion of red blood cells. The preferred unique α-N-acetylgalactosaminidases and α-galactosidases exhibit the following characteristics:
     (i) exclusive, preferred or no less than 10% substrate specificity for the type A and B branched polysaccharide structures relative to measurable activity with simple mono- and disaccharide structures and aglycon derivatives hereof; (ii) optimal performance at neutral pH with blood group oligosaccharides and in enzymatic conversion of cells; and (iii) a favorable kinetic constant K m  with mono- and oligosaccharide substrates. The conversion methods of the invention use significantly lower amounts of recombinant glycosidase enzymes than previous and result in complete sero-conversion of all blood group A and B red cells.

RELATED APPLICATIONS

This application claims the benefit of priority under 35 U.S.C. 119(e)to copending U.S. Provisional Application No. 60/324,970, filed on Sep.25, 2001, and No. 60/361,769, filed on Mar. 5, 2002, the entire contentsof which are incorporated herein by reference.

FIELD OF THE INVENTION

This invention relates to enzymatic removal of type A and B antigensfrom blood group A, B, and AB reactive cells in blood products, andthereby converting these to non-A and non-B reactive cells. Specificallythis invention relates to enzymatic removal of the immunodominantmonosaccharides specifying the blood group A and B antigens, namelyα1,3-D-galactose and α1,3-D-N-acetylgalactosamine, respectively. Moreparticularly, this invention relates to the use of uniqueα-N-acetylgalactosaminidases and α-galactosidases with superior kineticproperties for removal of the immunodominant monosaccharides of theblood group A and B antigens and improved performance in enzymaticconversion of red blood cells. Specifically, the preferred uniqueα-N-acetylgalactosaminidases and α-galactosidases exhibits the followingcharacteristics: (i) exclusive, preferred or no less than 10% substratespecificity for the type A and B branched polysaccharide structuresrelative to measurable activity with simple mono- and disaccharidestructures and aglycon derivatives hereof; (ii) optimal performance atneutral pH with blood group oligosaccharides and in enzymatic conversionof cells; and (iii) a favorable kinetic constant K_(m) with mono- andoligosaccharide substrates. This invention further relates to methodsfor use of these unique α-N-acetylgalactosaminidases andα-galactosidases in obtaining complete removal of A and B antigens oftype A, B, and AB cells determined by standard blood bank serologicaltyping and cross match analysis. More particularly, this inventionrelates to methods for conversion of cells using significantly loweramounts of recombinant glycosidase enzyme proteins than previously usedand obtaining complete sero-conversion of all blood group A and B redcells.

BACKGROUND OF THE INVENTION

As used herein, the term “blood products” includes whole blood andcellular components derived from blood, including erythrocytes (redblood cells) and platelets.

There are more than thirty blood group (or type) systems, one of themost important of which is the ABO system. This system is based on thepresence or absence of antigens A and/or B. These antigens are found onthe surface of erythrocytes and platelets as well as on the surface ofendothelial and most epithelial cells. The major blood product used fortransfusion is erythrocytes, which are red blood cells containinghemoglobin, the principal function of which is the transport of oxygen.Blood of group A contains antigen A on its erythrocytes. Similarly,blood of group B contains antigen B on its erythrocytes. Blood of groupAB contains both antigens, and blood of group O contains neitherantigen.

The blood group structures are glycoproteins or glycolipids andconsiderable work has been done to identify the specific structuresmaking up the A and B determinants or antigens. The ABH blood groupspecificity is determined by the nature and linkage of monosaccharidesat the ends of the carbohydrate chains. The carbohydrate chains areattached to a peptide (glycoprotein) or lipid (glycosphingolipid)backbone, which are attached to the cell membrane of the cells. Theimmunodominant monosaccharide determining type A specificity is aterminal α1-3 linked N-acetylgalactosamine (GalNAc), while thecorresponding monosaccharide of B type specificity is an α1-3 linkedgalactose (Gal). Type O cells lack either of these monosaccharides atthe termini of oligosaccharide chains, which instead are terminated withα1-2 linked fucose (Fuc) residues.

A great diversity of blood group ABH carbohydrate structures are founddue to structural variations in the oligosaccharide chains that carryABH immunodominant saccharides. Table 1 lists structures reported in manand those that have been found on human red cells or in blood extracts.For a review, see, Clausen & Hakomori, Vox Sang 56(1): 1-20, 1989). Redcells contain ABH antigens on N-linked glycoproteins andglycosphingolipids, while it is generally believed that O-linked glycanson erythrocytes glycoproteins, mainly glycophorins, are terminated bysialic acid and not with ABH antigens. Type 1 chain glycosphingolipidsare not endogenous products of red cells, but rather adsorbed fromplasma.

TABLE 1 Histo-Blood Group ABH Immunoreactive Determinants of HumanCells¹ Type of Found on Structure Name Hapten Structure GlycoconjugateRBC No A type 1, ALe^(d) GalNAcα1-3Galβ1-3GlcNAcβ1-R GlycolipidGlycolipid 1            2 N-linked        Fucα1 O-linked A type 1,ALe^(b) GalNAcα1-3Galβ1-3GlcNAcβ1-R Glycolipid Glygolipid 2           2         4 N-linked        Fucα1     Fucα1 O-linked A type2, A GalNAcα1-3Galβ1-4GlcNAcβ1-R Glycolipid Glycolipid 3            2N-linked N-linked        Fucα1 O-linked A type 2, ALe^(y)GalNAcα1-3Galβ1-4GlcNAcβ1-R Glycolipid Glycolipid? 4           2         3 N-linked        Fucα1     Fucα1 O-linked A type3, O-linked GalNAcα1-3Galβ1-3GalNAcα1-O-Ser/Thr 5            2       Fucα1 O-linked A type 3, RepetitiveGalNAcα1-3Galβ1-3GalNAcα1-3Galβ1-4GlcNAcβ1-R Glycolipid Glycolipid 6           2                2        Fucα1            Fucα1 A type 4,Globo GalNAcα1-3Galβ1-3GalNAcβ1-3Galα1-R Glycolipid Glycolipid? 7           2        Fucα1 A type 4, GanglioGalNAcα1-3Galβ1-3GalNAcβ1-3Galβ1-R Glycolipid 8            2       Fucα1 B type 1, BLe^(d) Galα1-3Galβ1-3GlcNAcβ1-R GlycolipidGlycolipid 9         2 N-linked     Fucα1 O-linked B type 1 BLe^(b)Galα1-3Galβ1-3GlcNAcβ1-R Glycolipid Glycolipid 10         2         4N-linked     Fucα1     Fucα1 O-linked B type 2, BGalα1-3Galβ1-4GlcNAcβ1-R Glycolipid Glycolipid 11         2 N-linkedN-linked     Fucα1 O-linked B type 2, BLe^(y) Galα1-3Galβ1-4GlcNAcβ1-RGlycolipid Glycolipid? 12         2        3 N-linked     Fucα1    Fucα1O-linked B type 3, O-linked Galα1-3Galβ1-3NAcα1-O-Ser/Thr 13         2    Fucα1 O-linked B type 4, Globo Galα1-3Galβ1-3GalNAcβ1-3Galα1-RGlycolipid? Glycolipid 14         2     Fucα1 B type 4, GanglioGalα1-3Galβ1-3GalNAcβ1-3Galβ1-R Glycolipid? 15         2     Fucα1 Htype 1, Le^(d)     Galβ1-3GlcNAcβ1-R Glycolipid Glycolipid 16      2N-linked Fucα1 O-linked H type 1, Le^(b)     Galβ1-3GlcNAcβ1-RGlycolipid Glycolipid 17      2 N-linked Fucα1 O-linked H type 1, H    Galβ1-3GlcNAcβ1-R Glycolipid Glycolipid 18      2 N-linked N-linkedFucα1 O-linked H type 2, Le^(y)    Galβ1-4GlcNAcβ1-R GlycolipidGlycolipid 19     2        3 N-linked Fucαl     Fucαl O-linked H type3,O-linked    Galβ1-3GalNAcα1-OSer/Thr 20     2 Fucαl O-linked H type 3,H-A    Galβ1-3GalNAcα1-3Galβ1-4GlcNAcβ1-R Glycolipid Glycolipid 21    2                2 (A RBC) Fucα1           Fucα1 H type 4, Globo   Galβ1-3GalNAcβ1-3Galα1-R Glycolipid Glycolipid 22     2 Fucα1 H type4, Ganglio    Galβ1-3GalNAcβ1-3Galβ1-R Glycolipid 23     2 Fucα1Thomsen-Friedenrich    Galβ1-3GalNAcα1-O-Ser/Thr O-linked O-linked 24Tf, T (+SA) Gal-A,    Galβ1-3GalNAcα1-3Galβ1-4GlcNAcβ1-R GlycolipidGlycolipid 25 T cross-react.                      2 (A RBC) Tn, Across-react.           GalNAcα1-O-Ser/Thr O-linked O-linked 26 (+SA)¹Adapted from Clausen and Hakomori, Vox Sang 56(1): 1–20, 1989.Designations: “?” indicates potential glycolipid structures which havenot been reported to date.

Blood group A and B exist in several subtypes. Blood group A subtypesare the most frequent, and there are three recognized major sub-types ofblood type A. These sub-types are known as A₁, A intermediate (A_(int))and A₂. There are both quantitative and qualitative differences thatdistinguish these three sub-types. Quantitatively, A₁ erythrocytes havemore antigenic A sites, i.e., terminal N-acetylgalactosamine residues,than A_(int) erythrocytes which in turn have more antigenic A sites thanA₂ erythrocytes. Qualitatively, A₁ erythrocytes have a dual repeated Astructure on a subset of glycosphingolipids, while A₂ cells have an Hstructure on an internal A structure on a similar subset of glycolipids(Clausen et al., Proc. Natl. Acad. Sci. USA 82(4): 1199-203, 1985,Clausen et al., J. Biol. Chem. 261(3): 1380-7, 1986). These differencesbetween A₁ and weak A subtypes are thought to relate to differences inthe kinetic properties of blood group A isoenzyme variants responsiblefor the formation of A antigens (Clausen et al., J. Biol. Chem. 261(3):1388-92, 1986). The differences of group B subtypes are believed to besolely of quantitative nature.

Blood of group A contains antibodies to antigen B. Conversely, blood ofgroup B contains antibodies to antigen A. Blood of group AB has neitherantibody, and blood group O has both. Antibodies to these and othercarbohydrate defined blood group antigens are believed to be elicited bycontinuous exposure to microbial organism carrying related carbohydratestructures. An individual whose blood contains either (or both) of theanti-A or anti-B antibodies cannot receive a transfusion of bloodcontaining the corresponding incompatible antigen(s). If an individualreceives a transfusion of blood of an incompatible group, the bloodtransfusion recipient's antibodies coat the red blood cells of thetransfused incompatible group and cause the transfused red blood cellsto agglutinate, or stick together. Transfusion reactions and/orhemolysis (the destruction of red blood cells) may result therefrom.

In order to avoid red blood cell agglutination, transfusion reactions,and hemolysis, transfusion blood type is cross-matched against the bloodtype of the transfusion recipient. For example, a blood type A recipientcan be safely transfused with type A blood, which contains compatibleantigens. Because type O blood contains no A or B antigens, it can betransfused into any recipient with any blood type, i.e., recipients withblood types A, B, AB or O. Thus, type O blood is considered “universal”,and may be used for all transfusions. Hence, it is desirable for bloodbanks to maintain large quantities of type O blood. However, there is apaucity of blood type O donors. Therefore, it is desirable and useful toremove the is immunodominant A and B antigens on types A, B and AB bloodin order to maintain large quantities of universal blood products.

In an attempt to increase the supply of type O blood, methods have beendeveloped for converting certain type A, B and AB blood to type O blood.Conversion of B cells to type O cells has been accomplished in the past.However, conversion of the more abundant A cells has only been achievedwith the less abundant weak A subgroup cells. The major obstacle fordevelopment and utilization of enzyme converted universal O cells has,in the past, been the failure to enzymatically convert the strong A₁cells. This obstacle has remained. As will be explained below in detailthe enzymes and methods used in the prior art are inefficient,impractical, and/or too costly to be used in a commercial process tosupply universal type O cells.

Conversion of B Cells:

Enzymatic conversion of type B blood using purified or recombinantcoffee bean (Coffea canephora) α-galactosidase has been achieved using100-200 U/ml (U.S. Pat. No. 4,427,777; Zhu et al., Arch Biochem Biophys1996; 327(2): 324-9; Kruskall et al., Transfusion 2000; 40(11): 1290-8).The specific activity of coffee bean α-galactosidase was reported to be32 U/mg using p-nitrophenyl α-D-Gal with one unit (U) defined as oneμmole substrate hydrolyzed per minute (Zhu et al., Arch Biochem Biophys1996; 327(2): 324-9). Enzymatic conversions were done at pH 5.5 withapproximately 6 mg/ml enzyme at 80-90% hematocrit, and the resultingconverted O cells functioned normally in transfusion experiments and nosignificant adverse clinical parameters were observed (Kruskall et al.,Transfusion 2000; 40(11): 1290-8). This data along with earlierpublications, clearly demonstrate that enzymatic conversion of red bloodcells is feasible and that such enzyme group B converted O (B ECO) cellscan function as well as matched type untreated cells in transfusionmedicine. Nevertheless, the quantities of enzymes used in these studies,even with present days most effective recombinant expression technology,renders ECO cells impractical mainly for economical reasons.

Claims of improved protocols for conversion of B cells using recombinantGlycine max α-galactosidase with a specific activity of approximately200 U/mg have been reported using 5-10 units/ml with 16% hematocrit(U.S. Pat. Nos. 5,606,042; 5,633,130; 5,731,426; 6,184,017). The Glycinemax α-galactosidase was thus used at 25-50 μg/ml, which represents asignificant reduction in enzyme protein quantities required (50-200fold) (Davis et al., Biochemistry and Molecular Biology International,39(3): 471-485, 1996). This reduction is partly due to the higherspecific activity of the Glycine max α-galactosidase (approximately 6fold) as well as different methods used for conversion and evaluation.The 200 U/ml enzyme used in the study of Kruskall et al., (Transfusion,40(11): 1290-8, 2000) was worked out for full unit (approximately 220 mlpacked cells) conversions at 80-90% hematocrits and thoroughly analyzedby standard blood bank typing as well as by more sensitive cross-matchanalysis. Furthermore, the efficiency of conversion was evaluated byanalysis of survival and induced immunity in patients receiving multipletransfusions of converted cells. The enzymatic conversions were done intest tubes in ml scale at 16% hematocrit, as described in U.S. Pat. No.5,606,042 (and U.S. Pat. No. 5,633,130; U.S. Pat. No. 5,731,426; U.S.Pat. No. 6,184,017) with Glycine max α-galactosidase, and the conversionefficiency not evaluated by cross-match analysis. Conversion of cells at16% hematocrit required 10 U/ml, while conversions at 8% required 5U/ml, indicating that converting at increased hematocrit requires moreenzyme although higher cell concentrations were not tested. Thus, partof the reduction in enzyme protein quantities required compared toprotocols reported by Kruskall et al., (Transfusion 2000; 40(11):1290-8), is related to the concentration (hematocrit) of cells used inconversion, and this may represent more than 5-10 fold although directcomparison is not possible without experimentation. The U.S. Pat. No.5,606,042 (and U.S. Pat. No. 5,633,130; U.S. Pat. No. 5,731,426; U.S.Pat. No. 6,184,017) further provides improvements in the conversionbuffer using Na citrate and glycine at less acidic pH (preferably pH5.8) and including additional protein in the form of BSA (bovine serumalbumin) for stabilization. Interestingly, the conversion bufferdeveloped for the Glycine max α-galactosidase was found not to beapplicable to coffee bean α-galactosidase. Although, some improvement inthe conversion of B cells may be provided by U.S. Pat. No. 5,606,042(and U.S. Pat. No. 5,633,130; U.S. Pat. No. 5,731,426; U.S. Pat. No.6,184,017), it is clear that at least more than 0.5 mg of enzyme isrequired per ml packed type B red cells using the disclosed protocol. Itis likely that considerable more enzyme than this is required to obtaincells fully converted to O cells by the most sensitive typing proceduresused in standard blood bank typing protocols. Furthermore, the protocolrequires introduction of additional extraneous protein (BSA or humanserum albumin) as well as exposing cells to acidic pH.

It is evident from the above that further improvements in conversion ofB cells is required in order to make this a practical and commerciallyapplicable technology. Necessary improvements include obtaining moreefficient alpha-galactosidase enzymes, which allow conversion to takeplace preferable at neutral pH and without extraneous protein added.

Conversion of A Cells:

Levy and Animoff (J. Biol. Chem. 255: 1737-42, 1980) tested the abilityof purified Clostridium perfringens α-N-acetylgalactosaminidase toconvert A cells, and found reduction in antigen expression butconsiderable blood group A activity remained. Further studies of thisenzyme have lead to purification to apparent homogeneity with a specificactivity using the αGalNAc p-nitrophenyl substrate of 43.92 U/mg (Hsiehet al., IUBMB Life, 50(2): 91-7, 2000; PCT Application No. WO 99/23210).The purified enzyme had a neutral pH optimum with the αGalNAcp-nitrophenyl substrate, but no studies of the activity of this enzymewith oligosaccharides were presented. Some degradation of the A₂ epitopewith the purified enzyme in an ELISA assay was reported, but the enzymehave not been evaluated in enzyme conversion of A₂ cells withappropriate blood typing.

Goldstein (Prog Clin Biol Res 165: 139-57, 1984; Transfus Med Rev 3(3):206-12, 1989) was unsuccessful in converting A cells using chicken liverα-N-acetylgalactosaminidase. U.S. Pat. No. 4,609,627 entitled “EnzymaticConversion of Certain Sub-Type A and AB Erythrocytes”, is directed to aprocess for converting A_(int) and A₂ (including A₂B erythrocytes) toerythrocytes of the H antigen type, as well as to compositions of type Berythrocytes which lack A antigens, which compositions, prior totreatment, contained both A and B antigens on the surface of saiderythrocytes. The process for converting A_(int) and A₂ erythrocytes toerythrocytes of the H antigen type, which is described in U.S. Pat. No.4,609,627, includes the steps of equilibrating certain sub-type A or ABerythrocytes, contacting the equilibrated erythrocytes with purifiedchicken liver α-N-acetylgalactosaminidase enzyme for a period sufficientto convert the A antigen to the H antigen, removing the enzyme from theerythrocytes and re-equilibrating the erythrocytes. U.S. Pat. No.6,228,631 entitled “Recombinant α-N-acetylgalactosaminidase enzyme andcDNA encoding said enzyme” provides a recombinant source for the chickenenzyme. The specific activities of purified and recombinant Pichiapastoris produced chicken liver α-N-acetylgalactosaminidase werereported to be approximately 51-56 U/mg using p-nitrophenyl αGalNAc assubstrate (Zhu et al., Protein Expression and Purification 8: 456-62,1996). The described conversion conditions for A_(int) and A₂ cells inU.S. Pat. No. 4,609,627 included 180 U/ml cells (hematocrit notspecified) at acidic pH 5.7, and treated cells did not agglutinate withunspecified anti-A reagent. This protocol requires more than 3 mg/mlenzyme protein and has not been reported to convert type A₁ cells.

Hata et al. (Biochem Int. 28(1): 77-86, 1992) also reported conversionof A₂ cells using chicken liver α-N-acetylgalactosaminidase at acidicpH. U.S. Pat. No. 5,606,042 (and U.S. Pat. No. 5,633,130; U.S. Pat. No.5,731,426; U.S. Pat. No. 6,184,017) disclose similar results.

Falk et al. (Arch Biochem Biophys 290(2): 312-91991, 1991) demonstratedthat an α-N-acetylgalactosaminidase purified from Ruminococcus torquesstrain IX-70 could destroy Dolichus biflorus agglutinability indicatingthat the A antigenic strength of A₁ cells was reduced to the level of A₂cells.

Izumi et al. (Biochem Biophys Acta 1116: 72-74, 1992) tested purifiedAcremonium sp. α-N-acetylgalactosaminidase on type A₁ cells. Althoughsome reduction in agglutination titer was observed using 7,000 U/ml (140U/20 μl) 4% hematocrit, conversion was not complete.

Human α-N-acetylgalactosaminidase enzyme has been isolated, cloned andexpressed (Tsuji et al., Biochem. Biophys. Res. Commun. 163: 1498-1504,1989, Wang et al., Human α-N-acetylgalactosaminidase-molecular cloning,nucleotide sequence, and expression of a full-length cDNA. Homology withhuman alpha-galactosidase A suggests evolution from a common ancestralgene. J. Biol. Chem. 265: 21859-66, 1990) (U.S. Pat. No. 5,491,075). ThepH optimum of human α-N-acetylgalactosaminidase is 3.5 (Dean K J,Sweeley C C. Studies on human liver alpha-galactosidases. II.Purification and enzymatic properties of alpha-galactosidase B(alpha-N-acetylgalactosaminidase). J. Biol. Chem. 254: 10001-5, 1979),similar to that of the human α-galactosidase (Dean K J, Sweeley C C.Studies on human liver alpha-galactosidases. I. Purification ofalpha-galactosidase A and its enzymatic properties with glycolipid andoligosaccharide substrates. J. Biol. Chem. 254: 9994-10000, 1979).

It is evident from the above that enzymatic conversion of type A cells,and particularly subgroup A₁ cells constituting up to 80% of group A,has not been accomplished to date. Therefore, there exists a need in theprior art to identify appropriate enzymes capable of converting group Acells by removing all immunoreactive A antigens. Furthermore, thereexists a need to develop appropriate conversion conditions preferably atneutral pH and without requirement of additional extraneous proteins.

Screening Assays:

Previous methods for searching, identification and characterization ofexo-glycosidases have generally relied on the use of simplemonosaccharide derivatives as substrates to identify saccharide andpotential linkage specificity. Derivatized monosaccharide, or rarelyoligosaccharide, substrates include without limitations p-nitrophenyl(pNP), benzyl (Bz), 4-methyl-umbrelliferyl (Umb), and7-amino-4-methyl-coumarin (AMC). The use of such substrates provideseasy, fast, and inexpensive tools to identify glycosidase activities,and makes large scale screening of diverse sources of enzymespractically applicable. However, the kinetic properties and finesubstrate specificities of glycosidase enzymes may not necessarily bereflected in assays with such simple structures. It is also possiblethat novel enzymes with high degree of specificity and/or selectiveefficiency for complex oligosaccharide and unique glycoconjugatestructures exists, but that these may have been overlooked and remainunrecognized due to methods of analysis. Thus, in order to identify andselect the optimal exo-glycosidase for a particular complexoligosaccharide or glycoconjugate structure it may be preferable to usesuch complex structures in assays used for screening sources of enzymes.Furthermore, assays used for screening may include selection forpreferable kinetic properties such as pH requirement and performance onsubstrates, e.g., attached to the membrane of cells.

In the prior art, all α-galactosidases (EC 3.2.1.22) andα-N-acetylgalactosaminidases (EC 3.2.1.49) used for destroying B and Aantigens of blood cells have been identified and characterized usingprimarily p-nitrophenyl monosaccharide derivatives. Interestingly, allα-galactosidase and α-N-acetylgalactosaminidase enzymes used in paststudies to attempt removal of A and B antigens on cells are evolutionaryhomologous as evidenced by significant DNA and amino acid sequencesimilarities. Thus, the human α-galactosidase andα-N-acetylgalactosaminidase are close homologues (Wang et al., J BiolChem, 265: 21859-66, 1990), and other enzymes previously used in bloodcell conversion including the chicken liver α-N-acetylgalactosaminidase,fungal acremonium α-N-acetylgalactosaminidase, and bacterialα-galactosidases all exhibit significant sequence similarities. Primarystructures of bacterial α-N-acetylgalactosaminidases have not beenreported in the scientific literature. Because these glycosidases sharesequence similarity it may be anticipated that the enzymes have relatedkinetic properties. Sequence analysis of all known O-glycosidehydrolases have been grouped in 85 distinct families based on sequenceanalysis, and the above mentioned α-galactosidases andα-N-acetylgalactosaminidases are grouped in families 27 and 36 (see,e.g., the webpage entitled “CAZy—Carbohydrate-Active Enzymes (FamilyGH32)” and located at http://afmb.cnrs-mrs.fr/˜cazy/CAZY/GH_(—)32.html).These enzymes are characterized by having a retaining mechanism ofcatalysis and use aspartic acid as the catalytic nucleophile (Henrissat,Biochem Soc Trans, 26(2): 153-6, 1998; Rye & Withers, Curr Opin ChemBiol, 4(5): 573-80, 2000).

Therefore, there exists in the art a need to identify newα-galactosidase and α-N-acetylgalactosaminidase activities andcorresponding enzyme proteins. If such enzymes exist, it is likely thatthey would not classify within families 27 and 36 because they would beselected to have significantly different kinetic properties.

SUMMARY OF THE INVENTION

The present invention provides compositions and methods for theenzymatic removal of type A and B antigens from blood group A, B, and ABreactive cells in blood products, and the conversion of these to non-Aand non-B reactive cells. Specifically, this invention providescompositions and methods for enzymatic removal of the immunodominantmonosaccharides specifying the blood group A and B antigens, namelyα1,3-D-galactose and α1,3-D-N-acetylgalactosamine, respectively.

The novel glycosidase enzymes of the present invention have beenspecifically selected for use in the removal of the immunodominantmonosaccharides, αGalNAc and αGal, from complex oligosaccharide targetsclose to the true A and B carbohydrate antigens of the surface of cellsin blood products. Preferred α-N-acetylgalactosaminidase enzymes of thisinvention have the following characteristics: (i) no less than 10%activity with blood group A oligosaccharides (tetrasaccharide or higher)compared to simple α-GalNAc monosaccharide derivatives; and (ii) activein red blood cell conversion at neutral pH (pH 6-8) with blood group Aoligosaccharides. The α-N-acetylgalactosaminidases of the presentinvention remove all detectable A antigens of all group A cells,including group A₁. Preferred α-galactosidase enzymes of this inventionhave the following characteristics: (i) no less than 10% activity withblood group B oligosaccharides (tetrasaccharide or higher) compared tosimple α-Gal monosaccharide derivatives; and (ii) active in red bloodcell conversion at neutral pH (pH 6-8) with blood group Boligosaccharides. The α-galactosidase enzymes of the present inventionhave no detectable activity with P₁ antigens. More preferredacetylgalactosaminidase and α-galactosidase enzymes are of bacterial orfungal origin, thereby permitting efficient and inexpensive recombinantexpression in prokaryotic and lower eukaryotic cells. In a preferredembodiment, the enzymes of this invention have no detectable activitywith p-nitrophenyl monosaccharide derivatives. In another preferredembodiment, enzymes of this invention have a favorable kinetic constantK_(m) with mono- and oligosaccharide substrates. A particularlypreferred α-galactosidase enzyme is further characterized as migratingin the 40-80 kD region by reducing SDS-PAGE analysis. Anotherparticularly preferred α-galactosidase enzyme comprises the amino acidsequence: Phe-Ala-Asn-Gly-Leu-Leu-Leu-Thr (SEQ ID NO: 1).

In another aspect, this invention provides methods for the completesero-conversion of all blood group A and B red cells, resulting in thecomplete removal of A and B antigens from type A, B, and AB cells. Theremoval of A and/or B antigens can be determined by standard blood bankserological typing or cross match analysis. According to the methods ofthis invention, the A and B antigens are removed using theα-N-acetylgalactosaminidases and/or α-galactosidases that (i) have noless than 10% activity with blood group A or B oligosaccharides(tetrasaccharide or higher) compared to simple mono- and disaccharidestructures and aglycon derivatives; and (ii) are active in red bloodcell conversion at neutral pH (pH 6-8). In a preferred embodiment, thesesero-conversion methods using significantly lower amounts of recombinantglycosidase enzyme proteins than methods known in the art. These methodscomprise the steps of: (a) contacting the blood product with the enzyme,under neutral pH conditions, for a period sufficient to remove theantigens, and (b) removing the enzyme from the blood product.

In one embodiment, this invention provides methods for the removal ofall detectable A antigens from group A or AB red cells, including groupA₁, using α-N-acetylgalactosaminidases that (i) have no less than 10%activity with blood group A oligosaccharides (tetrasaccharide or higher)compared to simple α-GalNAc monosaccharide derivatives; and (ii) areactive in red blood cell conversion at neutral pH (pH 6-8) with bloodgroup A oligosaccharides.

In another embodiment, this invention provides methods for the removalof all detectable B antigens from group B or AB red cells, usingα-galactosidases that (i) have no less than 10% activity with bloodgroup B oligosaccharides (tetrasaccharide or higher) compared to simpleα-Gal monosaccharide derivatives; and (ii) are active in red blood cellconversion at neutral pH (pH 6-8) with blood group oligosaccharides.

In yet another embodiment, this invention provides methods for theremoval of all detectable A and B antigens from group AB red cells usingan α-N-acetylgalactosaminidases and an α-galactosidases, each having oneor more of the following characteristics: (i) have no less than 10%activity with oligosaccharide structures (tetrasaccharide or higher)compared to simple mono- and disaccharide structures and aglyconderivatives; and (ii) are active in red blood cell conversion at neutralpH (pH 6-8) with blood group oligosaccharides.

In another aspect of the present invention, there are providedsero-converted erythrocytes. In one embodiment, the sero-convertederythrocytes are characterized as: (i) having been converted from a typeA or type AB erythrocyte to a non-A erythrocyte by anα-N-acetylgalactosaminidase; (ii) having A associated H structures; and(iii) having no detectable A antigens, including A₁ antigens, asdetermined by standard blood bank serological typing and cross matchanalysis. In another embodiment, the sero-converted erythrocytes arecharacterized as: (i) having been converted from a type B or type ABerythrocyte to a non-B erythrocyte by an α-galactosidase; (ii) havingretained P₁ antigenicity if of P₁ blood group; and (iii) having nodetectable B antigens, as determined by standard blood bank serologicaltyping or cross match analysis. In yet another embodiment, thesero-converted erythrocytes are characterized as: (i) having beenconverted from a type AB erythrocyte to a non-A, non-B erythrocyte by anα-N-acetylgalactosaminidase and an α-galactosidase; (ii) having Aassociated H structures; and (iii) having retained P₁ antigenicity if ofP₁ blood group; and (iii) having no detectable B antigens, as determinedby standard blood bank serological typing or cross match analysis.

In yet another aspect, this invention provides methods for the screeningand selection of enzymes with the above described preferred uniquecharacteristics and methods of purification and amino acid sequencinguseful for cloning and expression of the genes encoding these enzymes.These methods provide bacterial isolates producing such preferredenzymes.

In one embodiment, the method for screening and selecting anα-galactosidase enzyme useful for removing type B antigens from bloodgroup B and AB reactive cells in blood products under neutral pHconditions comprises the step of: (a) contacting a candidateα-galactosidase enzyme, under neutral pH conditions, with a group Boligosaccharide substrate and measuring the activity of the candidateenzyme with the group B oligosaccharide substrate; (b) contacting saidcandidate α-galactosidase enzyme, under neutral pH conditions, with anα-Gal monosaccharide derivative and measuring the activity of thecandidate enzyme with the group B monosaccharide derivative; and (c)comparing the relative activity of the candidate enzyme with the group Boligosaccharide substrate and α-Gal monosaccharide derivative.Candidates having no less than 10% activity with blood group Boligosaccharides (tetrasaccharide or higher) compared to simple α-Galmonosaccharide derivatives are selected as useful for removing type Bantigens from blood group B and AB reactive cells in blood productsunder neutral pH conditions.

In another embodiment, the method for screening and selecting anα-N-acetylgalactosaminidase enzyme useful for removing type A antigensfrom blood group A and AB reactive cells in blood products under neutralpH conditions comprises the step of:

(a) contacting a candidate α-N-acetylgalactosaminidase enzyme, underneutral pH conditions, with a group A oligosaccharide substrate andmeasuring the activity of the candidate enzyme with the group Aoligosaccharide substrate; (b) contacting said candidateα-N-acetylgalactosaminidase enzyme, under neutral pH conditions, with anα-GalNAc monosaccharide derivative and measuring the activity of thecandidate enzyme with the group A monosaccharide derivative; and (c)comparing the relative activity of the candidate enzyme with the group Aoligosaccharide substrate and α-GalNAc monosaccharide derivative.Candidates having no less than 10% activity with blood group Aoligosaccharides (tetrasaccharide or higher) compared to simple α-GalNAcmonosaccharide derivatives are selected as useful for removing type Aantigens from blood group A and AB reactive cells in blood productsunder neutral pH conditions.

BRIEF DESCRIPTION OF THE DRAWING

FIG. 1 illustrates the activity of recombinant coffee beanα-galactosidase with Galα-pNP at different pH. Assays were performed inreaction volumes of 0.5 ml containing 1.25 μmoles (2.5 mM) substrate.Reactions were incubated 20 min at 26° C., and quenched by addition ofan equal volume of 0.2 M sodium borate buffer (pH 9.8). Release ofp-nitrophenyl was quantified at OD 405 nm and plotted against pH.Buffers used: pH 5.0 and 5.5:20 mM NaOAc; pH 6.0-8.0:20 mM NaPO₄.

FIG. 2 illustrates the activity of recombinant coffee beanα-galactosidase with the blood group B tetrasaccharide AMC substrate atdifferent pH. Assays were performed in reaction volumes of 10 μlcontaining 1 nmole of substrate in Na Citrate-NaPO₄ buffers, pH 2.6-7.4and Na PO₄ buffer, pH 8.0. Reactions were incubated 40 min at 26° C., 3μl of the reaction mixture was spotted onto HPTLC and developed inCHCl₃:methanol:H₂O (60:35:8) and photographed. Panel A depicts the HPTLCanalysis, Std indicates migration of substrate without enzyme; Panel Bdepicts the substrate cleavage quantified by scanning and plottedagainst pH.

FIG. 3 illustrates the activity of recombinant coffee beanα-galactosidase with the Galili pentasaccharide substrate at differentpH. Assays were performed in reaction volumes of 10 μl containing 5nmoles of substrate in Na Citrate-NaPO₄ buffers. Reactions wereincubated 20 min at 26° C., 2 μl of each reaction mixture was spottedonto HPTLC and developed in CHCl₃:methanol:H₂O (30:60:20) and visualizedby orcinol spray. Panel A depicts the HPTLC analysis; Panel B depictsthe substrate cleavage quantified by scanning and plotted against pH.

FIG. 4 is an HPTLC analysis of five selected Streptomyceteα-galactosidase activities with the B tetrasaccharide AMC substrate.Designated strain numbers are the same as in Table V. The assay wasperformed as a time course with time points 20, 100 and 1000 minassessed by HPTLC. Migration of standard disaccharide, trisaccharide(H), and tetrasaccharide (B) AMC derivatives is indicated by arrows. NE,no enzyme control; Origin, sample application position. The appearanceof a disaccharide AMC product most pronounced in #2075 is due tocontaminating α-fucosidase activity.

FIG. 5 is an HPTLC analysis of five selected Streptomyceteα-galactosidase activities with Galα-pNP substrate. Assays wereperformed for 4 days at 30° C. Only strain #2260 showed significantactivity with the pNP substrate, and no galactose release at all wasdetected in the extract of strain #2357.

FIG. 5 b is an HPTLC analysis of five selected Streptomyceteα-N-acetylgalactosaminidase activities with the blood group Atetrasaccharide (Panel B-1) and A heptasaccharide AMC (Panel B-2)substrate. Streptomycete strains identified by numbers as in Table VII.Assays were performed in reactions of 10 μl containing 1 nmole ofAMC-substrates, 5.0 μl of selected fractions of the enzyme, and thebuffer 0.05M Na Citrate pH 6.0. Reactions were incubated 180 min at 30°C., and 2.5 μl of the reaction mixture was spotted onto HPTLC anddeveloped in CHCl₃:methanol:H₂O (60:35:8) and photographed. Migration oftrisaccharide (H), and tetrasaccharide (A), heptasaccharide andhexasaccharide (H-A) AMC derivatives is indicated by arrows. NE, noenzyme control; rCHl-Az, recombinant Chicken liver A-zyme; Origin,sample application position. The appearance of a disaccharide AMC is dueto contaminating α-fucosidase activity.

FIG. 6 is an Analysis of Streptomycete #2357 α-galactosidase enzymeseparated by S12 chromatography. Pre: sample before chromatography.Panel A: HPTLC analysis of activity with the B tetrasaccharide AMCsubstrate. Reactions were performed in volumes of 10 μl containing 1nmole substrate, 2 μl of the indicated S12 fractions, in 50 mM sodiumcitrate (pH 6.0) at 30° C. for 80 minutes. HPTLC was performed with 2.5μl and developed with CHCl₃:methanol:H₂O (60:35:8), dried andphotographed. The peak activity area containing contaminatingα-fucosidase activity in fractions 7-9 were pooled. Designations: Std, Btetrasaccharide AMC substrate without enzyme; Co, control reaction withcoffee bean α-galactosidase. Panel B: SDS-NuPAGE analysis of fractions.Designations as in Panel A.

FIG. 7 is an SDS-NuPAGE of pooled fractions from S12 chromatography ofenzyme activity purified from #2357. R-250 stained PVDF membrane ofSDS-NuPAGE. The protein band excised for sequencing is indicated by anarrow.

FIG. 8 is an HPTLC analysis of substrate specificities of recombinantcoffee bean α-galactosidase and purified α-galactosidase from #2357. Thecoffee bean α-galactosidase (lanes 1) efficiently cleaved all substratestested, while the purified α-galactosidase from #2357 selectively onlycleaved the blood group B tetrasaccharide. Panel A: Galili substrate(Galα1-3Galβ1-4GlcNAcβ1-3Galβ1-4Glc); Panel B: P^(k) substrate(Galα1-3Galβ1-4Glc-OGr); Panel C: P₁ substrate(Galα1-3Galβ1-4GlcNAc-OGr); and Panel D: B substrate(Galα1-3[Fucα1-2]Galβ1-4GlcNAcβ-OGr. HPTLC in Panels A, B and C weredeveloped using CHCl₃:methanol:H₂O (60:35:8), and Panel D inCHCl₃:methanol:H₂O (30:60:10).

FIG. 9 illustrates the activity of purified α-galactosidase from #2357with the blood group B tetrasaccharide AMC substrate at different pH.Assays were performed in reaction volumes of 10 μl containing 1 nmole ofsubstrate in 20 mM NaOAc (pH 5.0-5.5) or NaPO₄ (pH 6.0-8.0). Reactionswere incubated 40 min at 26° C., 3 μl of the reaction mixture wasspotted onto HPTLC and developed in CHCl₃:methanol:H₂O (60:35:8) andphotographed. Panel A-1: HPTLC analysis, Std indicates migration ofsubstrate without enzyme; Panel A-2: Substrate cleavage quantified byscanning and plotted against pH.

FIG. 9B illustrates analysis of purified α-galactosidase from #2357spiked with BSA separated by S12 chromatography. Panel B-1: SDS-NuPAGEanalysis of fractions 26-36. Designations: Mw: molecular weight markers;Pre: sample before chromatography. Panel B-2 HPTLC analysis of activitywith the B tetrasaccharide AMC substrate. Reactions were performed involumes of 10 μl containing 1 nmole substrate, 2 μl of the indicated S12fractions, in 50 mM NaPO4, pH 7.0. HPTLC was performed with 2 μl anddeveloped with CHCl₃:methanol:H₂O (60:35:8), dried and photographed.Designations: Std, B tetrasaccharide AMC substrate without enzyme; Co,control reaction with coffee bean α-galactosidase; D-AMC,disaccharide-AMC; Tri-AMC, trisaccharide-AMC; Tetr-AMC;tetrasaccharide-AMC.

FIG. 10 illustrates the activity of E. coli expressedα-N-acetylgalactosaminidase with the blood group A tetrasaccharide AMCsubstrate at different pH. Assays were performed in reactions of 10 μlcontaining 1 nmole of α-tetra, 0.05 μg enzyme, and the buffer NaCitrate-NaPO4 at varying pH 2.6-8.0. Reactions were incubated 40 min at26° C., and 3 μl samples analyzed by HPTLC. Panel A: HPTLC analysis;Panel B: Substrate cleavage quantified by scanning and plotted againstpH.

FIG. 11 illustrates the influence of buffer system on enzymaticconversion of A₂ cells using E. coli expressedα-N-acetylgalactosaminidase. Washed A₂ red cells were incubated with5-20 mU/ml α-N-acetylgalactosaminidase in the designated buffers at 25°C. (30% cell suspension), and conversion evaluated at 30 and 60 min byagglutination with Ortho anti-A.

FIG. 12 illustrates the influence of pH using 250 mM glycine buffer onenzymatic conversion of A₁ and A₂ cells using E. coli expressedα-N-acetylgalactosaminidase. Washed red cells were incubated with 7.5mU/ml α-N-acetylgalactosaminidase in 250 mM Glycine buffer pH 6.0 to 8.0at 25° C. (30% cell suspension), and conversion evaluated at 30 and 60min by agglutination with Ortho anti-A.

FIG. 13 illustrates the influence of glycine buffer concentration onenzymatic conversion of A₁ cells using E. coli expressedα-N-acetylgalactosaminidase. Washed red cells were incubated with 7.5mU/ml α-N-acetylgalactosaminidase in 100-400 mM Glycine buffer pH 7.0 at25° C. (30% cell suspension), and conversion evaluated at 30 and 60 minby agglutination with Ortho anti-A.

FIG. 14 illustrates the influence of concentration of E. coli expressedα-N-acetylgalactosaminidase on enzymatic conversion of A₁ and A₂ cells.Washed red cells were incubated with 5-50 mU/mlα-N-acetylgalactosaminidase in 250 mM Glycine pH 7.0 at 25° C. (30% cellsuspension), and conversion evaluated at 30 and 60 min by agglutinationwith Ortho anti-A.

FIG. 15 illustrates the influence of concentration of cells (hematocrit)on enzymatic conversion of A₁ cells using E. coli expressedα-N-acetylgalactosaminidase. Washed red cells were incubated with 20mU/ml α-N-acetylgalactosaminidase in 250 mM Glycine pH 7.0 at varyingconcentrations 20-90% at 25° C., and conversion evaluated at 30 and 60min by agglutination with Ortho anti-A.

FIG. 16 illustrates the influence of reaction time on enzymaticconversion of A₁ and A₂ cells using E. coli expressedα-N-acetylgalactosaminidase. Washed red cells were incubated with 5-50mU/ml α-N-acetylgalactosaminidase in 150 mM Glycine pH 7.0 at 25° C.(30% cell suspension), and conversion evaluated at 20, 40, 60, and 120min by agglutination with Ortho anti-A.

FIG. 17 illustrates the influence of temperature on enzymatic conversionof A₂ cells using E. coli expressed α-N-acetylgalactosaminidase. Washedred cells were incubated with 1-10 mU/ml α-N-acetylgalactosaminidase in200 mM Glycine pH 5.5 at 15° C., 25° C., and 37° C. (30% cellsuspension), and conversion evaluated at 20, 40, and 60 min byagglutination with Ortho anti-A.

DETAILED DESCRIPTION OF THE INVENTION

This invention is directed to the development and application of ascreening and selection strategy for novel α-N-acetylgalactosaminidasesand α-galactosidases with preferred specificities for the blood group Aand B structures and with preferred performance in enzymatic conversionof blood cells at neutral pH. Table 1 lists the complex structures of Aand B antigens found on blood cells. Quantitative studies of the kineticproperties of existing glycosidases with these complex structures havenot been reported. This is due partly to the difficulties in obtainingthese compounds from natural sources and partly because of thedifficulty and time-consuming efforts involved in synthesizing suchcomplex oligosaccharides by organic chemistry.

For the purpose of this invention, blood group A and B activeoligosaccharide AMC derivatives were synthesized (structures 3, 6, 11,25), and H variants hereof were either synthesized or produced byenzymatic removal of αGal or αGalNAc from the former structures.Furthermore, glycosphingolipids with structures 3, 6, 21, and 25 werepurified from human erythrocytes or produced therefrom by glycosidasetreatments as previously described (Clausen et al., Proc. Natl. Acad.Sci. USA 82(4): 1199-203, 1985, Clausen et al., J Biol Chem. 261(3):1380-7, 1986, Clausen et al., Biochemistry 25(22): 7075-85, 1986,Clausen et al., J. Biol. Chem. 262(29): 14228-34, 1987). Thin-layerchromatography assays to quantitatively determine removal of αGal orαGalNAc from the AMC derivatives or glycosphingolipids were developed.

Our initial analysis of the relative specific activities of recombinantcoffee bean α-galactosidase comparing activities with p-nitrophenylα-galacto side and a tetrasaccharide group B hapten substrate (structure11 AMC derivative) as representative of blood group B antigens revealeda striking difference of nearly 2000 fold. Thus, the coffee beanα-galactosidase had a specific activity of approximately 30-40 U/mg atpH 6.5 with p-nitrophenyl α-galactoside, as previously reported (Zhu etal., Arch Biochem Biophys 324: 65-70, 1995), but only 17 mU/mg with thetetrasaccharide group B substrate. This enzyme is therefore relativelyinefficient in destroying group B antigens, and an enzyme withpreference for the B tetrasaccharide is likely to exhibit much betterkinetic efficiency with group B structures.

Our initial analysis of the relative specific activities of recombinantchicken liver α-N-acetylgalactosaminidase comparing activities withp-nitrophenyl α-N-acetylgalactosamine and a tetrasaccharide group Ahapten substrate (structure 3 AMC derivative) as representative of bloodgroup A antigens again revealed a striking difference of over 100 fold.Thus, the chicken α-N-acetylgalactosaminidase as reported previously hada specific activity of approximately 50 U/mg at pH 3.65 withp-nitrophenyl α-N-acetylgalactosamine (Zhu et al., Protein Exp andPurification 8: 456-462, 1996), but only 0.2 U/mg at pH 5.5 (0.3 U/mg atpH 3.65) with the tetrasaccharide group A substrate. This enzyme istherefore relatively very inefficient in destroying group A antigens.

Since these two enzymes constitute state of the art performance inenzymatic conversion of blood cells, and that these either have failedto convert cells (group A) or are impractical due to enzyme quantitiesrequired (group B), in addition to both enzymes only performing in bloodcell conversion at acidic pH, it is clear that improved kineticproperties of enzymes for use in blood conversion are needed, and thatone likely strategy for initial identification is to analyze ratio ofactivities with p-nitrophenyl and complex A/B, substrates. Enzymes withpreferred or exclusive activities for the group A or B complexsubstrates are likely to perform more efficient in blood cellconversion.

Past difficulties in converting group A blood cells have mainly been dueto inability to convert the strong A₁ subgroup. As described above thegroup A₁ subgroup have more A antigens than other subgroups, but alsocontain a repeated A structure in the form of glycosphingolipids (Table1, structure 6). A₂ and possible weaker subgroups also contain an Aextended series of glycosphingolipids designated H-A and Gal-A (Table 1,structures 21 and 25), but these do not react with anti-A antibodies asoriginally described by Clausen et al., (Clausen et al., Proc. Natl.Acad. Sci. USA 82(4): 1199-203, 1985, Clausen et al., J Biol Chem.261(3): 1380-7, 1986, Clausen et al., Biochemistry 25(22): 7075-85,1986, Clausen et al., J. Biol. Chem. 262(29): 14228-34, 1987). This isfurther confirmed by the findings that extensiveα-N-acetylgalactosaminidase treated subgroup A₂ cells type as O withtyping reagents as described above. The difficulty in convertingsubgroup A₁ in comparison to subgroup A₂ may therefore be due to thequantitative difference in amounts of A antigens, the presence ofrepetitive A glycosphingolipids on A₁ cells only, or a combination ofthese. An important parameter of preferred α-N-acetylgalactosaminidasesfor use in group A conversion is the ability to efficiently cleave theterminal αGalNAc residue on repetitive A glycosphingolipids. Analysis ofthe efficiency of the recombinant chicken liverα-N-acetylgalactosaminidase revealed comparable specific activities(approximately 0.3 U/mg) with A type 2 tetrasaccharide AMC derivative(structure 3) and repetitive A type 3 AMC derivative (structure 6). Itmay be concluded from this that the failure of the chicken liverα-N-acetylgalactosaminidase to convert all group A cells is not due tothe unique A₁ structures. Furthermore, this data may indicate that Atetrasaccharides contain sufficient structure of the group A (and B)antigens to be used to determine the kinetic properties andspecificities of α-N-acetylgalactosaminidases, as well as for predictionof their performance in blood cell conversion.

Preferred α-N-acetylgalactosaminidases and α-galactosidases have neutralpH optima and can be produced cost-effectively as recombinant proteinsin unicellular organisms such as bacteria and yeast. The presentinvention developed a screening assay for the preferred enzymeactivities using A and B tetrasaccharide AMC derivative substrates andmeasuring activities at neutral pH. Further, activities were compared toactivities using p-nitrophenyl monosaccharide derivatives in order toidentify activities with preference or exclusivity for the complexsubstrates. Application of this screening assay on a large panel ofbacterial and fungal isolates (3100) identified several bacterialisolates expressing α-N-acetylgalactosaminidase or α-galactosidaseactivities measured with A or B tetrasaccharide AMC substrates, but noor insignificant levels of activity with the corresponding p-nitrophenylmonosaccharide substrates. One of each activities were further analyzedafter sero- and genotyping these as Streptomyces strains. Analysis ofstrain #8 determined to express α-N-acetylgalactosaminidase activityrevealed that the activity was insoluble and associated with the cellmass. Strain #8 was deposited on Feb. 14, 2002 with the American TypeCulture Collection (ATCC) and has been assigned ATCC Deposit No.PTA-4076. In contrast, strain #2357 determined to expressα-galactosidase activity revealed that the activity was soluble andfound in the supernatant of a French press. Strain #2357 was depositedon Feb. 14, 2002 with the American Type Culture Collection and has beenassigned ATCC Deposit No. PTA-4077. Because it is considerable simplerto purify a soluble protein, we chose to initially purify and sequencethe enzyme protein from #2357. The activity of #2357 was purified to aspecific activity of more than 10 U/mg with the B tetrasaccharidesubstrate, while no activity with p-nitrophenyl α-galactoside wasdetectable. SDS-PAGE analysis of the resulting preparation revealed 3-4protein bands in the 40-80 kD region. Gel filtration analysis of thepreparation showed activity migrating comparable to BSA indicating amolecular weight of 40-80 kD. A single short sequence was obtained:

-   -   Phe-Ala-Asn-Gly-Leu-Leu-Leu-Thr SEQ ID NO: 1.

Detailed analysis of the substrate specificity of the partially purifiedα-galactosidase activity demonstrated an unprecedented fine specificityfor the branched B blood group structure, and no linear structurescapped by α1-3 or α1-4 galactose residues were cleaved. Analysis of pHoptimum showed this to be 5.5 to 7.0. The identified α-galactosidaseactivity is therefore highly preferred over enzymes in the prior artwith respect to restricted substrate specificity, high specific activityfor group B structures, and pH optimum.

Preliminary analysis of the α-N-acetylgalactosaminidase activity of #8revealed similar properties, but linear structures were cleaved as well.Due to difficulties in purification it was not possible to assess thespecific activity of this enzyme, but even partially purifiedpreparations at 0.1 U/mg, showed no detectable activity with thep-nitrophenyl is monosaccharide derivative.

The finding that the two identified and partially characterizedactivities were similar in nature, and entirely different from anypreviously reported α-galactosidase and α-N-acetylgalactosaminidaseactivities, strongly suggested that a unique novel family of homologuesglycosidases was identified by the screening strategy employed.

We next embarked on assaying all commercially available α-galactosidasesand α-N-acetylgalactosaminidases using our selecting assay to determineif enzymes with the preferred specificity were available. Oneα-N-acetylgalactosaminidase (NEB α-N-acetylgalactosaminidase) wasidentified that exhibited relative high substrate specificity for Atetra- and heptasaccharide AMC derivatives compared to the simpleαGalNAc monosaccharide derivatives. This enzyme is disclosed by thesupplier (New England BioLabs Inc, catalog no. P0734B) to be derivedfrom a proprietary strain and expressed in E. coli, and its substratespecificity described as catalyzing the hydrolysis of terminal α-GalNAclinkages from oligosaccharides. Specifically, it is disclosed inmaterial supplied with the enzyme that the substrate specificity includep-nitrophenyl-α-D-N-acetylgalactosaminopyranoside (p-nitrophenylα-GalNAc) and A tetrasaccharide AMC substrate (structures 3-8). We havenot found additional information in the scientific literature orelsewhere as regards this enzyme. Analysis of the kinetic properties ofthis enzyme with our panel of substrates revealed that the enzyme has aspecific activity of approximately 0.25 U/mg with the A tetrasaccharideAMC substrate, and less than 2.5 U/mg with p-nitrophenyl αGalNAc.Furthermore, the enzyme has a broad pH optimum 6.0-8.0. Although, thisenzyme only exhibits a moderate preferential substrate specificity forthe A tetrasaccharide AMC substrate and the specific activity with thissubstrate is relatively low, this enzyme partly has the proposedproperties of an optimal enzyme to be used in blood cell conversions andit can be expressed in bacteria.

As described above the identified Streptomyces α-galactosidase has aspecific activity with the B tetrasaccharide substrate exceeding 10 U/mgand it functions at maximum velocity at neutral pH. The enzyme was,however, not available in quantities and of purity required forevaluation of its performance in blood cell conversion. The identifiedStreptomyces α-N-acetylgalactosaminidase was similarly not available.Since the NEB α-N-acetylgalactosaminidase has the same identifyingcharacteristics as the two identified Streptomyces activities, althoughthe specific activity is only approximately 0.25 U/mg with the Atetrasaccharide substrate, the availability of this in recombinant pureform allowed for evaluation of this new class of glycosidases in bloodcell conversions.

We therefore tested the performance of the NEBα-N-acetylgalactosaminidase in group A blood cell conversion, in orderto confirm that the proposed preferred properties ofα-N-acetylgalactosaminidases used in the above screening and selectionstrategy actually selected for enzymes with improved characteristics inenzymatic conversion of red blood cells. The NEBα-N-acetylgalactosaminidase showed remarkable efficiency in conversionof both A₁ and A₂ blood cells at neutral pH. Using a fixed hematocrit of30% in enzyme reactions, a number of parameters of the conversionprocess were analyzed. The preferable buffer system is 200-300 mMglycine at pH 6.5 to 7.5. Several additives may be added to thisincluding but without limiting 1-5 mM NaCl, 1-5 mM CaCl₂, 1-10 mMphosphate buffered citrate, 0.25 mM Trisodium citrate, and 0.1 to 10%polyethylene glycol (PEG) of varying molecular weights from 300 to10,000. Approximately 5 mU/ml NEB α-N-acetylgalactosaminidase convertedA₂ cells and approximately 20 mU/ml converted A₁ cells in 60 minutes(30% hematocrit) to cells typing as O with routine blood bankingreagents and procedures. Increased amount of enzyme used resulted indecreased time required for conversion. Converted cells reacted withanti-H reagents as O cells, and analysis of physical parameters ofconverted cells revealed no changes from untreated cells (methemoglobin,2,3DPG, ATP and Osmotic fragility). To the best of our knowledge, thisis the first example of enzymatic conversion of intact group A₁ cells tocells typing as O.

The quantity of E. coli expressed α-N-acetylgalactosaminidase requiredfor conversion of group A cells (5-20 mU/ml) is equivalent to 20-80μg/ml enzyme protein. This is a considerable improvement over amounts ofα-N-acetylgalactosaminidase used in the prior art to convert A₂ cells (3mg/ml). It is also an improvement compared to the quantities ofα-galactosidase used to convert B cells, whether it is the coffee beanα-galactosidase (6 mg/ml at 80% hematocrit) or the Glycine maxα-galactosidase (50 μg/ml at 16% hematocrit). Furthermore, theconversions with NEB α-N-acetylgalactosaminidase were performed atneutral pH, while all other conversions in the past have been done atacidic pH 4.5-5.8.

The performance of the E. coli expressed α-N-acetylgalactosaminidasetherefore clearly confirms that the properties of this proposed newclass of exo-glycosidases, as defined by the criteria set out above,have improved performance in A and B blood cell conversions.Furthermore, the identification and characterization of a Streptomycesα-galactosidase with over 40 fold higher specific activity for the bloodgroup B tetrasaccharide substrate compared to the specific activity ofthe NEB α-N-acetylgalactosaminidase for group A the tetrasaccharide,indicates that the Streptomyces enzyme may require more than 40 foldless protein in conversions, i.e., 0.5-2 μg/ml at 30% hematocrit inreactions. Conversion of a unit of packed blood cells (approximately 220ml) would thus require less than 0.35-1.4 mg/unit. With presentbacterial, yeast and fungal expression technologies, it is possible toproduce recombinant enzymes at 5-10 US$/mg. It is therefore evident thatenzymatic conversion of blood cells requires enzymes with thecharacteristics and performance of the ones provided by this invention.

Strains 8 and 2357 were both deposited on Feb. 14, 2002 with theAmerican Type Culture Collection and have been assigned ATCC DepositNos. PTA-4076 and PTA-4077, respectively. These deposits with the ATCCwere made under the terms of the Budapest Treaty on the InternationalRecognition of the Deposit of Microorganisms for the Purpose of) PatentProcedure. Applicants acknowledge their duty to replace the depositshould the depository be unable to furnish a sample when requested dueto the condition of the deposit before the end of the term of a patentissued hereon. Applicants also acknowledge their responsibility tonotify the ATCC of the issuance of such a patent, at which time thedeposit will be made available to the public. Prior to that time, thedeposit will be made available to the Commissioner of Patents under theterms of 37 C.F.R. §1.14 and 35 U.S.C. §112.

EXAMPLES General Methods Used

A series of complex blood group ABH oligosaccharide structures as7-amino-4-methyl-coumarin derivatives were custom synthesized by AlbertaChemical Research Council as listed in Tables II, III, and IV. Otherstructures were available from different suppliers (Sigma, CalbioChem,New England Biolabs). Enzymes were prepared as previously reported (Zhuet al., Protein Expr Purif. 8(4): 456-62, 1996, Zhu et al., Arch BiochemBiophys. 324(1): 65-70, 1995), or purchased from suppliers as indicated.All reagents used were of analytical grade or higher. Standard enzymeassays were performed as following with the different glycosidases:

Recombinant Coffee Bean α-Galactosidase Expressed in P. pastoris:

Assays with p-nitrophenyl monosaccharide derivatives were performed bytwo is procedures:

-   -   i) 1.25 μmol substrate in reaction mixtures of a total volume of        0.5 ml containing 50 mM sodium citrate and 20 mM sodium        phosphate (pH 5.5) was incubated at 26° C. for 10 min. Reactions        were quenched by adding an equal volume 0.2 M sodium borate        buffer (pH 9.8). The amount of the liberated p-nitrophenol was        determined by measuring the absorbance at 405 nm compared to a        standard curve of p-nitrophenol (0.01-0.15 μmole);    -   ii) 1.25 μmol substrate in reaction mixtures of a total volume        of 0.5 ml containing 50 mM sodium citrate and 20 mM sodium        phosphate (pH 5.5) was incubated at 26° C. Five μl aliquots were        taken at different time points (0′, 5′, 15′, 30′, 60′) to follow        product development. Product development was analyzed by high        performance thin-layer chromatography (HPTLC) in        chloroform-methanol-water (vol/vol/vol: 60/35/8) visualized by        orcinol staining.

Assays with Derivatized oligosaccharide substrates (AMC, OGr) wereperformed by the following procedure:

-   -   iii) One (AMC) or 5 (OGr) nmol substrate in reaction mixtures of        a total volume of 10 μl containing 50 mM sodium citrate (pH 6.0)        was incubated at 26° C. Aliquots of 2.5-3.0 μl were taken at        different time points (0′, 15′, 30′, 60′) to follow product        development. Product development was analyzed by HPTLC in        chloroform-methanol-water (vol/vol/vol: 60/35/8) and visualized        by UV or orcinol staining.

Assays with free oligosaccharide substrates were performed by thefollowing procedure:

-   -   iv) 5 nmol substrate was incubated in a 10 μl reaction volume        containing 50 mM sodium citrate (pH 6.0) at 26° C. or 30° C. for        30-180 min. Product development was analyzed by HPTLC in        chloroform-methanol-water (vol/vol/vol: 30/60/10) and visualized        by orcinol staining.

Assays to determine K_(m) for substrates were modified as follows:

-   -   v) The concentration of the αGal p-nitrophenyl substrate was        varied from 5.0 mM to 0.04 mM (5 pmol/10 μl) using 0.1-0.5 μg        enzyme.

Recombinant Chicken Liver α-N-Acetylgalactosaminidase Expressed in P.pastoris:

Assays with p-nitrophenyl monosaccharide derivatives were performed bytwo procedures:

-   -   vi) 1.25 μmoles of αGalNAc p-nitrophenyl monosaccharide was        incubated in a tube containing 50 mM sodium citrate 20 mM sodium        phosphate at pH 2.8 at 37° C. for 60 min. 5.0 μl aliquots were        taken at different time points (0′, 15, 30′, 60′) to follow the        kinetics of product development. Product development was        analyzed by high performance thin-layer chromatography (HPTLC)        in chloroform-methanol-water (vol/vol/vol: 60/35/8) stained with        p-anisaldehyde and visualized by UV.

Commercial Recombinant α-N-Acetylgalactosaminidase (New EnglandBiolabs):

-   -   vii) 0.5 mM as a starting concentration of αGalNAc p-nitrophenyl        monosaccharide derivative in a tube containing 0.05 M Sodium        phosphate buffer pH 7.0 was sequentially diluted 2 fold by        mixing it with an equal volume of buffer. 0.5 kg of enzyme was        added to each tube and incubated for 10 min at 37° C. The        reaction was quenched by adding an equal volume of 0.2 M sodium        borate buffer (pH 9.8). The amount of the liberated        p-nitrophenol was determined by measuring the absorbance at 405        nm.

Example 1 Characterization of Fine Substrate Specificities ofα-N-Acetylgalactosaminidases and α-Galactosidases Previously Used in A/BBlood Cell Conversions

To eliminate the B and A antigenic activities of red cells, the mostefficient exoglycosidases used in the past have been the coffee beanα-galactosidase and the chicken liver α-N-acetylgalactosaminidase,respectively. These enzymes have been studied extensively and theircharacteristics and performance in red cell conversion described in theliterature and in patent applications as referenced above.

(i) Specific Activity with Different Substrates (U/mg).

Table II lists reported specific activities of these enzymes withp-nitrophenyl monosaccharide derivatives. One unit is defined as theactivity converting one micromole of substrate in one minute under theoptimal assay conditions defined. Assays with p-nitrophenyl substrateswere evaluated at initial velocity with less than 10% of the substratesused.

TABLE II Specific activities of α-galactosidases andα-N-acetylgalactosaminidases with monosaccharide derivatives.Recombinant Recombinant Coffee Chicken Liver Glycine Max. SubstrateStructure Blood Group Bean α-galactosidase α-N-acetylgalactosaminidaseα-galactosidase (derivative) Specificity pH 6.5 pH 3.65 pH 6.5 Galα1-pNP— 32 U/mg¹ — 295.6 U/mg² GalNAcα1-pNP — — 50 U/mg³ — ¹Zhu et al., (1995)Arch Biochem Biophys 324: 65-70, ²Davis et al., (1996) Biochem Mol BiolInt 39: 471-85, ³Zhu et al., (1996) Protein Exp and Purfication 8:456-462.

In the present invention, similar results were obtained for recombinantpurified coffee bean α-galactosidase and the chicken liverα-N-acetylgalactosaminidase. Information of the specific activities witholigosaccharide substrates resembling the A and B antigens have not beenreported. This is likely due to limited availability of such compounds.In the present invention, complex A and B structures were synthesizedand analysis of the kinetic parameters of the enzymes with substratesmimicking the antigens as found on red cells was predicted to aid indefining criteria for selecting novel enzymes with better properties inred cell conversion.

As shown in Table III, analysis of the specific activities of the twoenzymes with the tetrasaccharide AMC derivatives were dramatically lowerthan the activities obtained with p-nitrophenyl monosaccharidederivatives.

TABLE III Specific activities of α-galactosidases andα-N-acetylgalactosaminidases with blood group active oligosaccharidederivatives¹. Recombinant Recombinant Coffee Bean Chicken Liver BloodGroup α-galactosidase α-N-acetylgalactosaminidase Substrate Structure(derivative) Specificity pH 5.5 pH 3.65 pH 5.5Galα1-3(Fucα1-2)Galβ1-4GlcNAc-AMC B 0.017 U/mg — —GalNAcα1-3(Fucα1-2)Galβ1-4Glc-AMC A — 0.5 U/mg 0.4 U/mgGalNAcα1-3(Fucα1-2)Galβ1- A — 0.5 U/mg 0.4 U/mg3GalNAcα1-3(Fucα1-2)Galβ1-4Glc-AMC ¹Specific activities were determinedas described under Examples using assays with approximately 50% and 100%final conversion of substrates evaluated at three time points (20, 40and 60 min).

The specific activity of recombinant coffee bean α-galactosidaseexpressed in yeast and purified to homogeneity showed 32 U/mg withGalα1-pNP (at optimum pH 6.5). However, the specific activity ofrecombinant coffee bean α-galactosidase was only 17 mU/mg (approximately2000 fold less) when measured with a blood group B tetrasaccharide-AMCsubstrate at the optimal pH used for enzymatic conversion of red cellswith this enzyme (pH 5.5) (Table III).

Similarly, recombinant α-N-acetylgalactosaminidases from chicken liverrevealed a strong preference for non-blood group A structures withhighest activity measured with the non-natural substrate GalNAcα1-pNP.The specific activity of recombinant chicken α-N-acetylgalactosaminidaseexpressed in yeast and purified to homogeneity showed approximately 50U/mg with GalNAcα1-pNP at the optimal pH of 3.65 (Table II), while only0.3 U/mg (166 fold less) was measured with a blood group Atetrasaccharide-AMC substrate at pH 3.65 (Table III). The specificactivity at pH 5.5 was lower at only 0.2 U/mg.

Similar results were found for the Acremonium sp., and Patella vulgataα-N-acetylgalactosaminidases (not shown).

(ii) K_(m) for Different Substrates.

Reported Michaelis-Menton constants K_(m) and V_(max) (determined fromLineweaver-Burk plots) of the coffee bean α-galactosidase and thechicken liver α-N-acetylgalactosaminidase with different substrates areshown in Table IV.

TABLE IV Apparent K_(m) and V_(max) of α-galactosidases andα-N-acetylgalactosaminidases with monosaccharide derivatives.Recombinant coffee bean Glycine Max. Recombinant Chicken Liverα-galactosidase α-galactosidase α-N-acetylgalactosaminidase SubstrateStructure pH 5.5, 26° C. pH 5.6, 26° C. pH 3.65, 37° C. (derivative)K_(m) V_(max) K_(m) V_(max) K_(m) V_(max) Galα1-pNP 363 μM¹ 46.9 U/mg¹n.d.² n.d.² — — GalNAcα1-pNP — — — — 827 μM³ 60.9 U/mg³ ¹Zhu et al.,(1995) Arch Biochem Biophys 324: 65-70), ²Vosnidou et al., Biochem MolBiol Int 46(1): 175-186, 1998, ³Zhu et al., (1996) Protein Exp andPurfication 8: 456-462. Designation: n.d., not determined.

In the present invention similar K_(m) values were obtained forrecombinant purified coffee bean α-galactosidase and the chicken liverα-N-acetylgalactosaminidase. These K_(m) values are relatively high andenzymes with 10 to 100 fold lower K_(m) would represent preferredcandidates for red cell conversions as near complete removal of antigensis predicted to be important.

Thus, the observed high K_(m)'s of these enzymes with all substratesappears to represent another reason for the poor performance of theseenzymes in conversion of red cells.

An α-N-acetylgalactosaminidase isolated to apparent homogeneity from R.torgues was reported to have a specific activity of 50 U/mg withGalNAcα-pNP and a K_(m) of 2-8 mM (Hoskins et al., J Biol Chem. 272(12):7932-9, 1997). Although, this enzyme appear to have a neutral pH optimumstudies so far has not been able to demonstrate efficient enzymaticconversion of group A cells (Hoskins et al., Transfusion. 41(7): 908-16,2001). It is likely that the poor performance of this enzyme is linkedto the extremely high K_(m).

(iii) pH Optima for Different Substrates.

The pH optima of coffee bean and Glycine max α-galactosidases have beenreported to be broad and include neutral pH. Assays to measure pH optimawere performed with the simple artificial α-Gal monoaccharidep-nitrophenyl derivative. Nevertheless, neither of these enzymesperforms in blood cell conversions at neutral pH and conversions haveonly successfully been obtained at pH 5.5 to 6.4 (see discussion above).In order to provide insight into this phenomenon, we analyzed the pHoptimum of the coffee bean enzyme with the p-nitrophenyl galactose andthe oligosaccharide substrates B tetrasaccharide and the Galilipentasaccharide. As shown in FIG. 1, the pH optimum with the simplemonosaccharide substrate was as reported previously broad with maximumactivity at 6.4. In contrast, the pH optimum with the B tetrasaccharidesubstrate was acidic with maximum at 3.5 to 5.0 as shown in FIG. 2.Furthermore, a similar low pH optimum was found for the cleavage of theGalili oligosaccharide as shown in FIG. 3.

The optimal pH with of the coffee bean enzyme with melibiose, raffinose,and stachyose has been reported to be low (between 3.6-4) (Zhu et al.,(1995) Arch Biochem Biophys 324: 65-70, Courtois and Petek (1966)Methods Enzymol 3: 565-571). This is in agreement with our findings forthe B and Galili Oligosaccharides, and suggests that the enzymegenerally have a low pH optimum with natural disaccharides andoligosaccharides.

It is likely that the pH optimum of the enzyme with the p-nitrophenylsubstrate is artificial and linked to the physical properties of theaglycan rather than reflecting the properties of the enzyme with naturalsubstrates. The data presented here therefore may provide an explanationfor the failure of this enzyme to perform in red cell conversion atneutral pH.

The chicken liver α-N-acetylgalactosaminidase was reported to have pHoptimum at 3.65 using GalNAcα-pNP as described above. Analysis of theinfluence of pH on this enzymes activity with the blood group Atetrasaccharide AMC substrate was in agreement with the reported dataand showed a pH optimum of 3.5-4.5 (not shown).

As described above the chicken liver α-N-acetylgalactosaminidase andcoffee bean α-galactosidase enzymes are members of a large homologousglycosidase gene family including the human lyzosomal enzymes. Lyzosomalenzymes generally function at acidic pH and all of these have beenreported to have acidic pH optima. It is therefore likely that otherhomologous enzymes with sequence similarities to this group share thischaracteristic feature of an acidic pH optimum. We therefore chose toscreen new sources for α-N-acetylgalactosaminidase and α-galactosidaseactivities using the appropriate substrates and neutral pH.

Example 2 Identification of α-N-Acetylgalactosaminidases andα-Galactosidases with Highly Preferential or Exclusive SubstrateSpecificity for the Blood Group A and/or B Blood Group Structures atNeutral pH.

In order to identify potential enzymes with preferred and/or exclusivespecificity for blood group A and B structures, a large panel of fungaland bacterial isolates were analyzed. A protocol for initial screeningwith the blood group A/B tetrasaccharide AMC derivatives as well as theGal/GalNAcα-pNP derivatives was developed. Briefly, preserved frozenstocks of cultures were inoculated onto YM slant cultures (tube size:1.8×18 cm), grown at 27° C. for 8 days, and the cultures (spores)harvested by washing down with 5 ml cryogen (10% glycerol+5% lactose),followed by maceration (strongly whirling with glass beads in thescrewed tube, 1.3×13 cm). One ml of the slant cultures were inoculatedto appropriate specific media for aerobic fermentation (25° C. forfungal cultivation and 28° C. for actinomycete cultivation) for 72˜96hours. Samples of 2.5 ml of each grown cultures were macerated in ascrewed tube (1.3×13 cm) containing about 8-10 glass beads (size=3 mmdiameter) by vortexing for 15 minutes, after which the pH was adjustedto 6.5 with citrate buffer and the macerated cultures frozen in tubes at−20° C. Frozen cultures were thawed and macerated again as above andcentrifuged at 2100×g for 15 minutes. The supernatants served as enzymesource for the initial assay. Samples of 10 μl were tested as follows:

Assays with Group A or B Tetrasaccharide AMC Substrates:

Reaction mixtures of 10 μl containing 50 mM sodium citrate (pH 6.5),0.25 nmol oligosaccharide AMC substrate, and 10 μl enzyme source asdescribed above were incubated at 30° C., and product development wasmonitored at different time intervals (20 min to 48 hours) by HPTLC.

Assays with P-Nitrophenyl Monosaccharide Substrates:

Reaction mixtures of 20 μl containing 50 mM sodium citrate (pH 6.5), 2-5mM monosaccharide pNP substrate and 10 μl enzyme source as describedabove were incubated at 30° C., and product development was monitored atdifferent time intervals (20 min to 24 hours) by OD405 nm or HPTLC.

Screen for α-Galactosidase Activities:

A total of 2400 isolates were screened and five strains with significantactivities with the group B tetrasaccharide AMC substrate wereidentified. These strains were selected for a small scale fermentation,which was processed by French press, (4)₂SO₄ precipitation, andseparation on Q-Sepharose. Further analysis of the pooled peaks ofactivity found in Q-Sepharose fractions revealed specific activitieswith the two substrates as listed in (Table VI).

TABLE VI Substrate Specificity of Five Identified Streptomycesα-Galactosidase Activities. Specific activities of Q-Sepharose peakfractions¹ Enzyme Source U/mg (Strain) Galα1-pNPGalα1-3(Fucα1-2)Galβ1-4GlcNAc-AMC Strain #2075 <0.02 0.004 Strain #2110<0.03 0.0007 Strain #2260 0.0009 <0.00003 Strain #2357 n.d. 0.075 Strain#2371 <0.005 0.0001 ¹Analysis of specific activities were determined inpooled active fractions from Q-sepharose chromatography. Purificationwas done from 60 ml of broth with protease inhibitors (PMSF, leupeptin,pepstain, EDTA) subjected to French pressing at 10,000 psi. Thispreparation was centrifuged at 13,000 × g for 30 minutes, andsupernatant fractionated by ammonium sulfate precipitation at 15% and50%. The 15-50% pellet was dissolved in 20 mM Tris (pH 7.5), andfiltered through a 0.45 μm filter. The clarified filtrate was loadedonto a 5 ml Pharmacia Hi-trap Q column and the proteins were eluted witha 0-0.15 M NaCl gradient. Designation: n.d., not determined.

The HPTLC analysis with group B tetrasaccharide AMC substrate of thefive candidate strains is shown in FIG. 4. The activities of the fivestrains cleaved the B tetrasaccharide AMC substrate with varying degreeto a product migrating as H trisaccharide AMC as well as in some casesto a disaccharide AMC derivative. The latter is due to contaminatingα-fucosidase activity.

Strains 2075 and 2357 expressed highest activities with the Btetrasaccharide substrate. Activities with the αGal p-nitrophenylsubstrate did not correlate with the activities with the Btetrasaccharide substrate. During purification, it was further confirmedthat the two activities could be separated indicating that they werederived from different proteins. Only strain 2357 completely lackedactivity with the αGal p-nitrophenyl substrate, which made furtheranalysis simpler and this activity was chosen for further purificationand characterization. A small scale fermentation of #2357 was performedand the enzyme activity was found in the soluble fraction after Frenchpress (See Table VI legend).

Serotyping of strain #2357 by colony morphology was performed byAccugenix, Newark, Del., confirming it as an actinomycete. Genotyping byShort Tandem Repeats of 500 base pairs placed strain #2357 in the Genusof Streptomyces griseoplanus with 1.60% difference.

Screen for α-N-Acetylgalactosaminidase Activities:

A total of four strains with significant activities with the group Atetrasaccharide AMC substrate were identified (Table IV).

TABLE VII Substrate Specificity of Four Identified Streptomycesα-N-acetylgalactosaminidase Activities. Specific activities ofQ-Sepharose peak fractions¹ Enzyme U/mg Source GalNAcα1-3(Fucα1-2)GalNAcα1-3(Fucα1-2)Galβ1-3GalNAcα1- (Strain) GalNAcα1-pNPGalβ1-3GalNAc-AMC 3(Fucα1-2)Galβ1-4GlcNAc-AMC Strain 8 n.d. 0.00370.0037 Strain 1488 <0.00005 0.016 0.016 Strain 1647 n.d. 0.0055 0.0055Strain 2233 <0.00005 0.00028 0.00028 ¹Purification and assay asdescribed in legend to Table VI.

The HPTLC analysis with group A tetrasaccharide AMC substrate of thefour candidate strains is shown in FIG. 5 b.

All identified strains with significant activities with the Atetrasaccharide substrate showed none or barely detectable levels ofactivities with the p-nitrophenyl derivative.

Strains 8, 1488, and 1647 expressed the highest activities with the Atetrasaccharide substrate, but only the activity in #8 was stable andcould be recovered for further characterization. This isolate was chosenfor further analysis. A small fermentation was performed and the enzymeactivity found to be insoluble and associated with the pelleted fractionafter French press.

Serotyping of strain #8 by colony morphology was performed by Accugenix,Newark, Del., confirming it as an actinomycete. Genotyping by ShortTandem Repeats of 500 base pairs placed strain #8 in the Genus ofStreptomyces chattanoogensis with 0.00% difference.

The above data showed that bacteria contain α-galactosidase andα-N-acetylgalactosaminidase exoglycosidases with unique substratespecificities for the immunodominant αGalNAc or αGal residues of thecomplex blood group A and B antigens. Such enzymes are proposed to bepreferred for use in enzymatic blood cell conversions due to theirhighly preferred or exclusive specificities for the substrate as foundon red cells.

Example 3 Isolation and Characterization of a Novel α-GalactosidaseIdentified from Streptomyces Strain #2357, which has Exclusive SubstrateSpecificity for the Branched Blood Group B Antigens and withUnprecedented High Specific Activity with Such Substrates

A 20-liter fermentation culture was processed by the French pressmethod. The main α-galactosidase activity was determined to be presentin the supernatant after centrifugation at 10,000×g. The supernatant wasfractionated by ammonium sulfate precipitation and approximately 70%activity was found in the 20-60% fraction. The precipitate of the 20-60%cut was dissolved in 20 mM Tris (pH 7.5) and clarified bycentrifugation. The supernatant was sequentially fractionated bychromatography on Q-sepharose (buffer 20 mM Tris, pH 7.5, with agradient of 0-1.5 M NaCl), S-sepharose (buffer 20 mM NaOAc, pH 5.3, witha gradient of 0-1.0 M NaCl), and by S12 gel filtration chromatography(buffer 20 mM NaOAc, pH 5.3, with 0.5 M NaCl or 20 mM NaPO₄, pH 6.5,with 0.5 M NaCl). Enzyme activity with the B tetrasaccharide AMCsubstrate was monitored in fractions collected throughout thispurification scheme. Lack of activity with the Galα-pNP was confirmedthroughout the separation steps. The final purified enzyme activity wasrecovered in fractions of the S12 chromatography eluting correspondingto a molecular weight of approximately 70,000 similar to the elution ofbovine serum albumin run as a standard (FIG. 6, panel A). SDS-NuPAGEanalysis of the S12 chromatography fractions revealed multiple bands infractions containing α-galactosidase activity, but the fraction withpeak activity only contained a few bands migrating in the region of40-80 kD (FIG. 6, panel B).

The specific activity of the pooled enzyme peak from the last S12chromatography step was approximately 10 U/mg (protein determined bysilver staining of SDS-NuPAGE and comparing the desired protein bandwith the amount of protein in the protein bands in the molecular weightmarker). Comparing the elution of activity with that of bovine serumalbumin revealed that the activity eluted after BSA, which providesevidence that the active protein has a molecular size lower than BSA,i.e. lower than 65 kd, as evaluated by gel filtration chromatography(FIG. 9 b).

The pooled fractions from the S12 chromatography containing the peakactivity were further purified by reverse phase chromatography using aC4 column (BioRad) (buffer: 0.1% TFA with a gradient of 0-100%acetonitrile). Eluted proteins were analyzed by SDS-NuPAGE and thefractions contained most of the desired protein band migrating at 70 kDwere pooled and dried under vacuum. The pooled fraction was rerun onSDS-NuPAGE and blotted onto PVDF membrane and stained with R-250 (FIG.7). The desired protein band was excised and subjected to N-terminalsequencing using Applied Biosystems Model 494 Precise Protein Sequencerw/Model 140C Microgradient Delivery System and Model 785A ProgrammableAbsorbance Detector. A single short sequence was obtained:

-   -   Phe-Ala-Asn-Gly-Leu-Leu-Leu-Thr (SEQ ID NO: 1).

Since the isolated α-galactosidase activity was not purified tohomogeneity it is possible that the obtained sequence originates fromanother protein. Further purification is required to isolate andcharacterize the novel enzyme protein and the encoding gene, and this isin progress.

Nevertheless, the novel α-galactosidase activity was highly purified andhad a specific activity of over 10 U/mg with the B tetrasaccharide. Theenzyme preparation allowed detailed studies of the substrate specificityand kinetic properties of the novel enzyme. The substrate specificity ofthe purified #2357 α-galactosidase was characterized using a large panelof oligosaccharides and derivatives with terminal α-Gal residues. Theassay was performed as described above using 1-4 nmoles substrate andthe amount of enzyme required to cleave this amount of the Btetrasaccharide AMC structure in 60 min. HPTLC analysis was performed atdifferent time points. An example of the analysis is shown in FIG. 8.The substrate specificity of the purified #2357 α-galactosidase activityis summarized in Table VIII.

TABLE VIII Substrate Specificity of α-Galactosidases PurifiedRecombinant Streptomyces Blood Group Coffee Bean #2357 SubstrateStructure (derivative) Specificity α-galactosidase α-galactosidaseGalα-Mu − +¹ − Galα-pNP − + − GalNAcα-pNP − − − Galα1-3Galβ-OGr − + −Galα1-4Gal P + − Galα1-4Galβ1-4GlcNAcβ-OGr P_(l) + −Galα1-4Galβ1-4Glcβ-OGr P^(k) + − Galα1-3(Fucα1-2)Galβ-OGr B + +Galα1-3(Fucα1-2)Gal-AMC B + − Galα1-3(Fucα1-2)Galβ1-3GlcNAcβ-OGr B + +Galα1-3(Fucα1-2)Galβ1-3GalNAcα-OGr B + +Galα1-3(Fucα1-2)Galβ1-3GalNAcβ-OGr B + + Galα1-3(Fucα1-2)Galβ1-4Glc-AMCB + + Galβ1-3GalNAcβ1-3Galβ1-4Glc-AMC Tβ − −GalNAcα1-3(Fucα1-2)Galβ1-4Glc-AMC A − −Galα1-3Galβ1-4GlcNAcβ1-3Galβ1-4Glc Galili B + −Galα1-3(Fucα1-2)Galβ1-3(Fucα1-4)GlcNAcβ-OGr B + +Galα1-3(Fucα1-2)Galβ1-4(Fucα1-4)GlcNAcβ-OGr B + + ¹Designations: “+”:Cleavage was detected within 60 minutes, “−”: No cleavage was detectedby overnight incubation. The linear trisaccharide as well as Galili Bcleavage reactions were evaluated by HPTLC using CHCl₃:methanol:H₂O(30:60:10). All other cleavage reactions were analyzed usingCHCl₃:methanol:H₂O (60:35:8).

For comparison recombinant coffee bean α-galactosidase was included inall analyses. In agreement with our studies described in Example 1, thecoffee bean α-galactosidase showed activity with all structurescontaining a terminal α-Gal residue. Both cal-3 (blood group B and the“Galili-epitope” without fucose) and α1-4 (blood group P₁ and P^(k))were substrates and the length or branching of the oligosaccharidestructure only had effect on relative activity, i.e., the quantities ofenzymes required to reach completion (specific activities onlydetermined for Galα p-nitrophenyl and B tetrasaccharide AMC).

In striking contrast the activity identified and purified fromStreptomyces strain #2357 only exhibits activity with the blood group Bstructures when presented as a tetrasaccharide or longer. The inabilityof this enzyme to cleave p-nitrophenyl or methyl-umbrelliferylmonosaccharide αGal derivatives showed that the lack of activity withmonosaccharides are not simply due to the aglycan and conjugation. Thetrisaccharide structure, Galα1-3(Fucα1-2)Gal-AMC, was inactive which maybe related to the conjugation chemistry as the corresponding structure,Galα1-3(Fucα1-2)Galβ-OGr, served as a substrate. Except for this theStreptomyces α-galactosidase efficiently utilized all the branched groupB related structures, which represents all know B structures found onred cells (Table I). This is the first α-galactosidase exhibiting uniquesubstrate specificity for the blood group B structures and showing noactivity with the human blood group antigen P₁ as well as the rareantigen P^(k) Thus, enzymatic conversion of red cells with theStreptomyces α-galactosidase will result in intact P₁ antigenicity incontrast to treatments with known α-galactosidases including the coffeebean α-galactosidase (Kruskall et al. Transfusion 2000; 40(11): 1290-8).Similarly, it is expected that the rare P^(k) antigen will be intactafter enzymatic conversion. Approximately 80% of caucasian populationexpress the P₁ antigen on red cells and, although the function of thisantigen is unknown, it is considered an important improvement in theenzymatic conversions to limit the removal of antigens solely to the Aand B blood group antigens.

The pH optimum of the purified Streptomyces α-galactosidase was analyzedas shown in FIG. 9. The enzyme activity with B tetrasaccharide AMCsubstrate had a broad pH optimum around 5.5-7.0. This enzyme thereforeis expected to perform in red cell conversions at neutral pH in contrastto enzymes used in the past.

This is the first α-galactosidase or α-N-acetylgalactosaminidaseactivity identified that have exclusive or even preferred substratespecificity for the blood group B or A structures over simplemonosaccharide derivatives. The α-galactosidase enzyme has a specificactivity with the blood group B structures higher than 10 U/mg, which ismore than 500 fold higher than that measured for the coffee beanα-galactosidase, as described in Example 1. Although this information isnot available for all other identified and characterizedα-galactosidases, it is likely that these show the same poor propertiesas the coffee bean α-galactosidase, because they generally functionefficiently with the αGal p-nitrophenyl derivative and because the genesencoding these are homologous. The identified Streptomycesα-galactosidase in the present invention is therefore unique and withoutprecedence in the prior art, and the kinetic properties identified forthis enzyme holds great promise for performance in enzymatic B bloodcell conversion.

Example 4 Characterization of Recombinant α-N-AcetylgalactosaminidaseExpressed in E. coli

New England BioLabs Inc. has recently commercialized a recombinantα-N-acetylgalactosaminidase (catalog no. P0734B) disclosed to beexpressed in E. coli. The enzyme is derived from a proprietary strain,and reportedly catalyzes the hydrolysis of terminal α-GalNAc linkagesfrom oligosaccharides and αGalNAc p-nitrophenyl (New England BioLabsInc. catalog information). In a screen of commercially availableexo-glycosidases we found this α-N-acetylgalactosaminidase to partlyexhibit the preferred characteristic of having a relative high specificactivity with A tetra- and heptasaccharide AMC derivatives compared toαGalNAc monosaccharide derivatives (Table IX). Importantly, the absolutespecific activity with the blood group A derivatives of this enzyme isnot considerably different from that of, e.g., the chicken liver enzyme.However, the relative activity compared to the monosaccharide substrateis considerably different. Thus, the data suggests that the E. coliexpressed α-N-acetylgalactosaminidase has a better relative specificityfor the blood group A antigen.

TABLE IX Specific activity of E. coli expressedα-N-acetylgalactosaminidase. Recombinant Substrate α-N- Structure BloodGroup acetylgalactosaminidase (derivative) Specificity pH 5.5 pH 7.0Galα1-pNP — — — GalNAcα1-pNP —  2.3 U/mg¹  2.5 U/mg Galα1-3(Fucα1-2)- B— — Galβ1-4GlcNAcβ-AMC GalNAcα1-3(Fucα1-2)- A 0.27 U/mg  0.27 U/mgGalβ1-3GalNAcβ-AMC GalNAcα1-3(Fucα1-2)Galβ1- A 0.26 U/mg  0.27 U/mg3GalNAcα1-3(Fucα1-2)- Galβ1-4GlcNAcβ-AMC ¹Assay conditions were asfollows: Assays with p-nitrophenyl were done in reaction volumes of 0.5ml containing 0.05 μmoles (100 μM), 50 mM sodium phosphate (pH 5.5 or7.0), and 0.5 μg enzyme. Reactions were incubated 10 min at 37° C., andquenched by addition of an equal volume of 0.2 M sodium borate buffer(pH 9.8). Assays with AMC substrates were done in reaction volumes of 10μl containing 1 nmol substrate (100 μM), 50 mM sodium phosphate or 0.25M glycine (pH 5.5 or 7.0), and 0.05-0.1 μg enzyme. Reactions wereincubated at 26° C. or 37° C. and analyzed by HPTLC at time points 0,15, 30, and 60 min. Protein quantification was performed bysemi-quantification using Coomassie stained SDS-PAGE analysis andweighed BSA as comparator.

Analysis of the fine substrate specificity of the E. coli expressedα-N-acetylgalactosaminidase revealed that it similarly to the chickenliver α-N-acetylgalactosaminidase utilized blood group A and repetitiveA structures equally efficient (Table IX).

Further analysis with a panel of non-fucosylated oligosaccharidestructures with terminal αGalNAc residues showed that the enzyme hasapproximately equal efficiency with these substrates compared to thegroup A branched substrates (Table X).

TABLE X Substrate specificity of E. coli expressedα-N-acetylgalactosaminidase. Recombinant Substrate Blood Groupα-N-acetylgalactosaminidase Structure (derivative) Specificity pH 6.0and pH 7.0 GalNAcα1-2Galβ1-OGr − +¹ GalNAcα1-3Galβ1-4- − + GlcNAcβ1-OGrGalNAcα1-4Galβ1-4- − + Glcβ1-OGr GalNAcα1-3Galβ1-3- − + GlcNAcβ1-OGrGalNAcα1-3(Fucα1-2)- A + Galβ1-3GalNAcα1-OGr ¹Assays were performed inreaction mixtures of 10 μl containing 1-4 mnoles substrate (100-400 μM),50 mM sodium citrate (pH 6.0), and 0.125 μg enzyme. Reactions wereincubated at 31° C. and analyzed by HPTLC at time points 0, 30, 60 and120 min.

The E. coli expressed α-N-acetylgalactosaminidase exhibited a broad pHoptimum including pH 6.0-7.0 with both monosaccharide andoligosaccharide substrates FIG. 10. At acidic pH below 5.5 the activitydrops rapidly and at pH 4.4 and lower activity is hardly detectable.This is the first α-N-acetylgalactosaminidase identified with thepreferred neutral pH optimum characteristic for red cell conversion.

The α-N-acetylgalactosaminidase activity was largely unaffected bybuffer type: 50-250 mM glycine, 0.1 M glycylglycine, 20-50 mM sodiumphosphate, 12.5-25.0 mM sodium citrate, 12.5-25.0 sodium citrate and5.0-10.0 sodium phosphate, McIlvine solution pH 5.5, PBS, MES. Theenzyme was also unaffected by NaCl (0-150 mM), glutathione andn-octyl-β-D-glucopyranoside.

Finally, evaluation of the kinetic constant K_(m) for the monosaccharidederivative revealed that the enzyme has a significantly lower apparentK_(m) (Table XI), as compared to the chicken liverα-N-acetylgalactosaminidase or the α-galactosidases described in Example1 (Table IV).

TABLE XI Apparent Km and V_(max) of E. coli expressedα-N-acetylgalactosaminidase with monosaccharide derivatives. SubstrateRecombinant α-N-acetylgalactosaminidase Structure pH 7.0, 37° C.(derivative) K_(m) V_(max) Galα1-pNP — — GalNAcα1-pNP 10-50 μM 3.3 U/mg³¹Assay conditions were as follows: Assays with p-nitrophenyl were donein reaction volumes of 0.5 ml containing from 3.9-50 nmoles (1.5-100 μM)50 mM sodium phosphate (pH 5.5 or 7.0), and 0.5 μg enzyme. Reactionswere incubated 10 min at 37° C., and quenched by addition of an equalvolume of 20 mM sodium borate buffer (pH 9.8). The amount of theliberated p-nitrophenol was determined by measuring the absorbance at405 nm compared to a standard curve of p-nitrophenol. Michaelis-Mentenconstants K_(m) and V_(max) determined from Lineweaver-Burk plots. ³Zhuet al., (1996) Protein Exp and Purification 8: 456-462.

Furthermore, preliminary results indicate that the K_(m) for the bloodgroup A oligosaccharide substrates similarly is approximately 20 μM. Theassay used for this determination involved densitometric scans of thesubstrate/product ratio using the tetrasaccharide AMC substrate(GalNAcα1-3(Fucα1-2)Galβ1-4GalNAc-AMC). This assay is unreliable at lowconcentrations, and it is therefore possible that the K_(m) is evenlower than 20 μM.

In summary, the E. coli expressed α-N-acetylgalactosaminidase exhibits arelatively high preference for blood group A substrates, maximumactivity with blood group A substrates at neutral pH, and favorablekinetic properties defined by a low K_(m).

Example 5 Enzymatic Conversion of A₁ and A₂ Red Blood Cells to OPhenotype Cells Using E. coli Expressed α-N-Acetylgalactosaminidase, asEvaluated by Routine Typing Protocols

Complete removal of the immunodominant A epitopes on human group A redcells have not previously been reported as described in detail above.Enzymatic conversion of blood group A cells of the weak subgroup A₂ havebeen reported using the chicken liver α-N-acetylgalactosaminidase atacidic pH, but the results of conversions were not verified by sensitivetyping reagents and methods used in standard blood typing procedures. Asdetailed below in Table XII, initial attempts to improve the performanceof the chicken liver α-N-acetylgalactosaminidase using differentreaction conditions failed to produce completely converted cells. Whilereactivity with a monoclonal anti-A antibody from Dako could beabolished for A₂ cells, typing with more sensitive reagents clearlyrevealed that the enzymatic degradation of group A epitopes wereincomplete.

TABLE XII Conversion¹ of A₁ and A₂ red blood cells with recombinantchicken liver α-N-acetylgalactosaminidase Pre Enzyme Treatment PostEnzyme Treatment Ortho Dolichos Ulex Dako Ortho Dolichos Ulex DakoAnti-A biflorus europaeus Anti-A Anti-A biflorus europacus Anti-A A1Donor #1 4+ 4+ 0   3+ 4+ 0 4+ 3+ A1 Donor #2 4+ 4+ 0   4+ 4+ 0 4+ w+  A2Donor #1 4+ 4+ A2 Donor #2 4+ 0   3+ 3+ 4+ 0 4+ 1+ A2 Donor #3 4+ 0   4+4+ 4+ 0 4+ 0   ¹Protocols used for conversion with chickenα-N-acetylgalactosaminidase: Three conversion protocols were evaluatedfor conversion of A₁ and A₂ red cells with recombinant chicken liverα-N-acetylgalactosaminidase. Conversion Protocol - A A₂ red cells (BethIsrael Deaconess Medical Center, Boston, MA) drawn in EDTA tubes andstored at 4° C. for up to seven days, were washed three times in PBS(Phosphate Buffered Saline, pH 7.4), and resuspended to 10% in asolution of PBS and 7.5% PEG (pH 7.4). Cells were treated withrecombinant chicken liver α-N-acetylgalactosaminidase (100 U/ml) at 30°C. for 180 min while shaking. Cells were washed three times in 0.9%saline and resuspended to 3-5% in saline for typing. ConversionProtocol - B A₁ red cells (Beth Israel Deaconess Medical Center, Boston,MA) drawn in EDTA tubes and leukoreduced A₂ red cells (American RedCross, New England Region, Dedham, MA) were frozen in Glycerolyte57,(Baxter Healthcare Corporation, Fenwal Division: Deerfield, IL)according to the AABB Technical Manual, 13^(th) edition, Method 6.6 andstored at −70° C. Prior to enzyme treatment cells were deglycerolizedusing 9.0%saline, 2.5% saline, and 0.9% saline (Method 125 ofImmunohematology Methods by the American Red Cross was followed),resuspended to a hematocrit of 50% in a solution of PBS and 7.5% PEG (pH7.4) and recombinant chicken liver α-N-acetylgalactosaminidase (200U/ml) added. Reactions were incubated at 37° C. shaking for 4 hours,followed by three washes in 0.9% saline, and final suspension to 3-5% insaline for typing. Conversion Protocol - C Origin and storage of cellssame as described under protocol B. Deglycerolized red cells were washedtwice in PCI (pH 5.5) with 150 mM NaCl and resuspended to a hematocritof 50% in PCI (pH 5.5) with 150 mM NaCl. Cells were treated withrecombinant chicken liver α-N-acetylgalactosaminidase (200 U/ml) at 37°C. shaking for 4 hours, followed by three washes in 0.9% saline, andfinal suspension to 3-5% in saline for typing.

It is evident from the data in Table XII that apparent removal of Aantigens is achieved, when defined by one particular anti-blood group Aspecific monoclonal antibody that is not approved for blood typingprocedures (DAKO). A large number of such antibodies exist and, due tospecificity and low affinity binding, they are inappropriate forserological typing purposes. Development of monoclonal cocktails for ABOroutine typing to substitute previously used polyclonal antibodyreagents was a major achievement for the blood bank industry in 1990s.Analysis of removal of A antigens by these highly sensitive and approvedroutine typing reagents showed, in contrast to the DAKO, antibody thatlittle conversion had occurred, as defined by agglutination titer.Details of the typing assay used in this example is as follows:

-   -   Approved typing reagents used in hemagglutination assays were        murine monoclonal antibodies and plant lectins obtained from        Ortho Clinical Diagnostics, Raritan, N.J.; Gamma        Biologicals/Immucor, Norcross, Ga. Non-approved reagents        included murine monoclonal anti-A antibody from Dako and a panel        of monoclonal antibodies to blood group A variants produced        by H. Clausen (Clausen et al., Proc. Natl. Acad. Sci. USA 82(4):        1199-203, 1985, Clausen et al., J Biol Chem. 261(3): 1380-7,        1986, Clausen et al., Biochemistry 25(22): 7075-85, 1986,        Clausen et al., J Biol Chem. 262(29): 14228-34, 1987). Typing        reagents were used according to the manufacturers        recommendations and other monoclonal antibodies as determined by        titrations.        Hemagglutination Assay (Room Temperature)        1. A 3-5% suspension of washed red cells in isotonic blood bank        saline was prepared.        2. One drop (approx 50 μl) of antibody/lectin reagent was added.        3. One drop (approx 50 μl) of the red cell suspension was added        4. Tubes were mixed and centrifuged for 15 seconds at 3500 rpm.        5. Cells were resuspended by gentle agitation and examined        macroscopically for agglutination.        6. The agglutination was graded according to Method 1.8 in the        AABB Technical Manual, 13 edition.

Similar results were obtained with a purified fungalα-N-acetylgalactosaminidase from acremonium sp. (Calbiochem) (notshown).

As described in the previous examples, preferred enzymes for use inremoving blood group A or B epitopes from red cells are likely to haveparticularly good kinetic properties with oligosaccharide substratesresembling the blood group A or B antigens. Such preferred kineticproperties could be represented by preferred or exclusive substratespecificities for the blood group A or B oligosaccharides, and low or noactivity with simple monosaccharide derivatives such asmonosaccharide-pNP substrates. Preferred kinetic properties could alsobe represented by a particularly low K_(m) for relevant substrates.Further preferred kinetic properties consist of neutral pH optimum ofreactions with relevant blood group active substrates, and otherreaction conditions that are compatible with the integrity and functionsof red cells. Other preferred properties of the enzyme such as size,charge, solubility, and other physico-chemical properties may alsorelate to performance in enzymatic conversion of red cells.

Novel α-galactosidases and α-N-acetylgalactosaminidases with improvedkinetic properties were identified from various bacterial strains in thepresent invention as described in Examples 2, 3 and 4. Theα-N-acetylgalactosaminidase (New England Biolabs) described in Example 4represents one example of such an α-N-acetylgalactosaminidase and it wasavailable in recombinant form of sufficient purity to test ourhypothesis that enzymes with the above mentioned preferredcharacteristics would exhibit superior performance in red cellconversions.

Shown in Table XIII is the performance of thisα-N-acetylgalactosaminidase in red blood cell conversions at neutral pH.The α-N-acetylgalactosaminidase was capable of completely convertingboth A₁ and A₂ red blood cells to cells typing as O as defined byroutine blood bank typing protocols.

TABLE XIII Conversion of A₁ and A₂ red blood cells with NEBα-N-acetylgalactosaminidase Pre Enzyme Treatment Post Enzyme TreatmentOrtho Dolichos Ulex Ortho Gamma Dolichos Ulex Anti-A biflorus europaeusAnti-A Anti-A biflorus europaeus A1 Donor #1 4+   4+ 0   0 0 0 4+ A1Donor #2 4+   4+ 0   0 0 0 4+ A1 Donor #3 4+   4+ 0   0 0 0 4+ A2 Donor#1 4+ 0 3+ 0 0 0 4+ A2 Donor #2 4+ 0 3+ 0 0 0 4+ A2 Donor #3 4+ 0 2+ 0 00 4+ A2 Donor #4 4+ 0 3+ 0 0 0 4+ A2 Donor #5 4+ 0 2+ 0 0 0 4+ A2 Donor#6 4+ 0 3+ 0 0 0 4+ Protocol: Leuko-reduced red blood cells (OklahomaBlood Institute) or red cells collected from volunteers (ACD), werewashed once in 0.9% saline and resuspended in the conversion buffer to30% hematocrit. Cells were treated with 10 to 20 mU/ml (One unit isdefined as the amount of enzyme that hydrolyses 1 μmol of Atetrasaccharide AMC in 1 min using the standard reaction conditionsdescribed elsewhere) α-N-acetylgalactosaminidase (New England Biolabs)and incubated at 25° C. for 60 min with mixing. Treated cells werewashed once with 0.9% saline, resuspended to 3-5% in saline, and typedas described above.

Red cells of both A₁ and A₂ subtypes treated with 10-20 mUα-N-acetylgalactosaminidase at neutral pH were totally unreactive withthe anti-A typing reagents in direct agglutination assays. Insteadenzyme treated A cells became equally reactive as control O cells withthe lectin Ulex Europaeus, which is generally used as an anti-H reagent.The reactivity with Dolichus Biflorus which is generally used as ananti-A₁ reagent was destroyed within the first minutes of the treatment(not shown).

The cross-match analysis of α-N-acetylgalactosaminidase treated cellsshown in Table XIV confirmed that both A₁ and A₂ enzyme converted cellsbehaved as O control cells.

TABLE XIV Cross-match analysis (IS, immediate spin) of converted A₁ andA₂ red blood cells with NEB α-N-acetylgalactosaminidase IS of PostEnzyme Treatment of Red cells A₁ A₁ A₁ A₂ A₂ A₂ O Donor #1 Donor #2Donor #3 Donor #1 Donor #2 Donor #3 Donor #1 Plasma 0 0 0 0 0 0 0 Salinecontrol A₁ plasma (n = 2) 0 0 0 0 0 0 0 A₂ plasma (n = 2) 0 0 0 0 0 0 0O plasma (n = 7) 0 0 0 0 0 0 0

This result shows that O and B individuals, who have variable titers ofantibodies directed against blood group A antigens of A red cells, donot recognize these when the immunodominant αGalNAc residue issufficiently removed. As described in the Background of the Inventionand further illustrated in Table I, this result indicates that the minoramounts of glycosphingolipids containing the repetitive blood group Astructure in agreement with our analysis in Example 4 is fully convertedto the H associated A structure (Table I, structure 21). Furthermore, itindicates that this H associated A structure is perceived as a normal Hantigen by the immune system. This is in accordance with our previousstudies of the immunogenicity of this glycolipid antigen in mice(Clausen et al., J Biol Chem. 261(3): 1380-7, 1986, Clausen et al., JBiol Chem. 261(3): 1388-92, 1986). The finding that enzymatic digestionwith a single α-N-acetylgalactosaminidase enzyme renders A₁ as well asA₂ red cells non-reactive with anti-A typing reagents and plasma ofgroup O and B individuals is novel and a major advancement in developinga commercially viable technology for providing universally acceptableenzyme converted O cells. While enzyme converted B cells chemically arepredicted to be identical to O cells, enzyme converted A cells willphenotype as O but have two different types of H antigens. The majorityof these two being the H type 2 structure (Table I, structure 18) foundon O cells, but also a minor amount of H glycolipids with an internalstructure consisting of a masked A trisaccharide is present (Table I,structure 21). Single enzyme α-N-acetylgalactosaminidase converted Acells are hence distinct from O cells and any red cells previouslyprepared and used in transfusion medicine, however, they are expected tofunction identical to O cells.

Detailed studies of the parameters of enzyme conversion of red cellswith the E. coli expressed α-N-acetylgalactosaminidase were carried outfor optimization. While pH influenced the activity of the enzymeactivity with the A tetrasaccharide AMC substrate, none of theparameters tested and described below influenced this activitysignificantly.

Buffer System:

As shown in FIG. 11, the optimal buffer system appeared to be 250 mMglycine. Reactions in NaP and PCI buffers, which are generally used forenzymatic conversion of B cells, did not produce significant conversion.

Glycine Buffer pH:

The E. coli expressed α-N-acetylgalactosaminidase was found in Example 4to have a broad pH optimum around neutral pH. Analysis of the pH optimumin enzymatic conversion of A₁ and A₂ cells revealed a more definedoptimum at pH 7 (FIG. 12). Conversion of the weak A₂ cells was achievedat a broader range of pH 6-8 with 7.5 mU/ml enzyme, but if less enzymewas used the optimum was at pH 7 (not shown).

Glycine Buffer Molarity:

The concentration of glycine was found to be an important parameter forenzyme conversion of group A cells with the E. coli expressedα-N-acetylgalactosaminidase (FIG. 13). Optimal conversion was achievedat 250-300 mM.

Enzyme Concentration:

FIG. 14 illustrates titration of the E. coli expressedα-N-acetylgalactosaminidase from 5-50 mU/ml with A₁ and A₂ cells. Inagreement with A₁ having more A antigenic Epitopes than A₂, more enzymeis required to convert A₁ cells. Titration of enzyme on A₂ cells from1-10 mU revealed that 3 mU/ml was required to fully convert with theused conditions (not shown).

Influence of Concentration of Cells (Hematocrit) During Treatment:

Treatment of A₁ cells at concentrations from 20-90% with constant amountof enzyme (20 mU) showed that conversion efficiency decreased withincreasing cell concentration (FIG. 15). At higher concentrations ofenzyme, conversion occurred faster, but conversion efficiency at cellconcentrations above 50% did not improve proportionally suggestion thatoptimal conversion conditions are 20-50%.

Influence of Treatment Time:

FIG. 16 illustrates that conversion is proportional with amount ofenzyme and time.

Influence of Temperature:

The activity of the E. coli expressed α-N-acetylgalactosaminidase withsaccharide derivative at the temperature interval 20-40° C. was found tobe similar, and the performance of the enzyme in group A cell conversionas illustrated in FIG. 17 confirmed this.

These results clearly demonstrate that one α-N-acetylgalactosaminidase,exemplified by the E. coli expressed α-N-acetylgalactosaminidase used inthis example having the preferred unique kinetic properties defined inthis invention, exhibits improved performance in enzymatic conversion ofgroup A cells. Conversion of group A₁ cells, which has not previouslybeen achieved, was achieved with the preferred enzyme at neutral pH andat enzyme protein concentrations much below those previously used forconverting A₂ and B cells. The amount of enzyme used (10-20 mU/mlequivalent to 30-60 μg/ml) for conversion of 30% suspension of cells(hematocrit), is lower than any amount of enzyme reported in the priorart to enzymatically convert A and B red cells.

A₁ and A₂ cells treated with an exo-N-acetylgalactosaminidase as the E.coli expressed α-N-acetylgalactosaminidase used in this example capableof cleaving GalNAc from all blood group A structures will expose theclassical H type 2 chain antigens (structure 18, Table 1) as found onblood group O cells, but it will also leave a small amount ofglycolipids with the A associated H structure (structure 21, Table 1).Studies with monoclonal antibodies specifically reactive with H type 2(BE2) and H type 3 (HH14, MBr-1) (see Clausen et al., J Biol Chem.261(3): 1380-7, 1986) revealed as expected thatexo-N-acetylgalactosaminidase treated A cells reacted strongly with BE2and weaker with HH14 and MBr-1 (not shown). Since none of the anti-Aantibodies including those used for routine blood typing reacted withtreated cells (Table XIII) the α-associated H glycolipid structure isnot recognized as an A antigen. This was further confirmed bycross-match analysis (Table XIV). This is in agreement with the factthat anti-H type 3 chain antibodies fails to distinguish between theabove glycolipid and the structures named H-Globo and mucin-type H(structures 22 and 21, respectively, Table 1) (Clausen et al., J BiolChem. 261(3): 1380-7, 1986). Thus, althoughexo-N-acetylgalactosaminidase treated A cells behave as O cellsphenotypically, they differ structurally from O cells by having minoramounts of the unique H glycolipid antigens. The group A enzymeconverted cells typing as group O therefore constitute a novel entitywhich is highly useful as a universal transfusable type of blood.

The novel Streptomyces enzymes defined in Example 3 have properties 30fold or better compared to the α-N-acetylgalactosaminidase used in thisexample, and this and other enzymes with similar properties arepredicted to perform correspondingly better in enzymatic red cellconversions.

EQUIVALENTS

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, numerous equivalents to thespecific procedures described herein. Such equivalents are considered tobe within the scope of the present invention and are covered by thefollowing claims. Various substitutions, alterations, and modificationsmay be made to the invention without departing from the spirit and scopeof the invention as defined by the claims. Other aspects, advantages,and modifications are within the scope of the invention. The contents ofall references, URLs, issued patents, and published patent applicationscited throughout this application are hereby incorporated by reference.The appropriate components, processes, and methods of those patents,applications and other documents may be selected for the presentinvention and embodiments thereof.

1. A method for removing immunodominant α-galactose monosaccharides fromblood group B or AB reactive cells in a blood product, said methodcomprising the steps of: (a) contacting said blood product with anα-galactosidase enzyme under pH conditions ranging from 6 to 8, therebycleaving the immunodominant α-galactose monosaccharides from the B or ABreactive cells, (b) removing said enzyme and the immunodominantα-galactose monosaccharides from said blood product, and (c) assayingthe enzyme contacted blood product for serotype B or AB reactivity,wherein a lack of B or AB serotype indicates removal of theimmunodominant monosaccharides, wherein said α-galactosidase enzyme hasthe following characteristics: (i) active in red blood cell conversionat neutral pH, and (ii) isolated and purified from a non-recombinantstrain of Streptomyces griseoplanus, wherein said α-galactosidase enzymeis isolatable by a method comprising the steps of: (1) fermentationculturing of an α-galactosidase producing Streptomyces griseoplanusstrain; (2) disrupting the cultured Streptomyces griseoplanus strain ofstep (1); (3) isolating an α-galactosidase-containing supernatantfraction from the disrupted Streptomyces griseoplanus strain of step (2)by centrifugation; (4) treating the α-galactosidase-containingsupernatant fraction of step (3) with ammonium sulfate to yield a 20 to60 percent ammonium sulfate fraction enriched in the α-galactosidase;(5) purifying the α-galactosidase from the 20 to 60 percent ammoniumsulfate fraction of step (4) by anion exchange chromatography followedby cation exchange chromatography to yield an ion exchange purifiedα-galactosidase; and (6) fractionating the ion exchange purifiedα-galactosidase of step (5) by size-exclusion chromatography to yield apurified α-galactosidase which elutes with a molecular weight in therange of 40-80 kD.
 2. A method for converting type B or AB erythrocytesto non-B erythrocytes, said method comprising the steps of: (a)contacting said blood product with an α-galactosidase enzyme, under pHconditions ranging from 6 to 8, thereby cleaving the immunodominantα-galactose monosaccharides from the B or AB reactive cells, (b)removing said enzyme and the immunodominant α-galactose monosaccharidesfrom said blood product, and (c) assaying the enzyme contacted bloodproduct for serotype B or AB reactivity wherein a lack of B or ABserotype indicates removal of the immunodominant monosaccharides,wherein said α-galactosidase enzyme has the following characteristics:(i) active in red blood cell conversion at neutral pH, and (ii) isolatedand purified from a non-recombinant strain of Streptomyces griseoplanus,wherein said α-galactosidase enzyme is isolatable by a method comprisingthe steps of: (1) fermentation culturing of an α-galactosidase producingStreptomyces griseoplanus strain; (2) disrupting the culturedStreptomyces griseoplanus strain of step (1); (3) isolating anα-galactosidase-containing supernatant fraction from the disruptedStreptomyces griseoplanus strain of step (2) by centrifugation; (4)treating the α-galactosidase-containing supernatant fraction of step (3)with ammonium sulfate to yield a 20 to 60 percent ammonium sulfatefraction enriched in the α-galactosidase; (5) purifying theα-galactosidase from the 20 to 60 percent ammonium sulfate fraction ofstep (4) by anion exchange chromatography followed by cation exchangechromatography to yield an ion exchange purified α-galactosidase; and(6) fractionating the ion exchange purified α-galactosidase of step (5)by size-exclusion chromatography to yield a purified α-galactosidasewhich elutes with a molecular weight in the range of 40-80 kD.
 3. Themethod of claim 1, wherein the strain of Streptomyces griseoplanus is S.griseoplanus, ATCC Deposit No. PTA-4077.
 4. The method of claim 2,wherein the strain of Streptomyces griseoplanus is S. griseoplanus, ATCCDeposit No. PTA-4077.